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Supplement: 6^th Amino Acid Assessment Workshop: SESSION 3

Pharmacokinetics and Safety of Arginine Supplementation in Animals^1–3,

⁴ Department of Animal Science, ⁵ Department of Veterinary Physiology and Pharmacology, and ⁶ Department of Statistics, Texas A&M University, College Station, TX, 77843; ⁷ Department of Animal and Food Sciences, Texas Tech University, Lubbock, TX 79409; and ⁸ Department of Systems Biology and Translational Medicine, Texas A&M Health Science Center, College Station, TX 77843

^* To whom correspondence should be addressed. E-mail: g-wu{at}tamu.edu.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Anticipating the future use of arginine to enhance fetal and neonatal growth as well as to treat diabetes and obesity, we performed studies in pigs, rats, and sheep to determine the pharmacokinetics of orally or i.v. administered arginine and the safety of its chronic supplementation. Our results indicate that all 3 species rapidly catabolized the supplemental arginine. The elevated circulating concentrations of arginine generally returned to baseline levels within 4–5 h after administration, with the rates varying with the age and physiological status of the animals. The clearance of arginine was greater in pregnant than in nonpregnant animals, in young than in adult animals, in lean than in obese animals, and in type-1 diabetic than in nondiabetic animals. I.v. administration of arginine-HCl to pregnant ewes (at least 0.081 g arginine·kg body weight^–1·d^–1) did not result in any undesirable treatment-related effect. Neonatal pigs, growing-finishing pigs, pregnant pigs, and adult rats tolerated large amounts of chronic supplemental arginine (e.g. 0.62, 0.32, 0.21, and 2.14 g·kg body weight^–1·d^–1, respectively) administered via enteral diets without the appearance of any adverse effect. On the basis of the comparative studies and a consideration of species differences in food intake per kilogram body weight, we estimate that a 70-kg human subject should be able to tolerate long-term parenteral and enteral supplemental doses of 6 and 15 g/d arginine, respectively, in addition to a basal amount of arginine (4–6 g/d) from regular diets.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Emerging evidence shows that dietary supplementation with L-arginine (as arginine-HCl) is beneficial for enhancing the reproductive performance of pigs with naturally occurring intrauterine growth retardation (9), enhancing protein deposition and postnatal growth of milk-fed piglets (10), normalizing plasma glucose levels in streptozotocin-induced diabetic rats (11), reducing fat mass in obese Zucker diabetic fatty (ZDF) rats (12), and improving vascular function in diabetic rats (13). Therefore, arginine supplementation may offer a new approach to the prevention and treatment of fetal growth restriction, obesity, and diabetes, which are all major health problems in humans worldwide (14,15). Anticipating the future use of arginine to solve these problems related to developmental biology and nutrient metabolism, we have undertaken studies in pigs, rats, and sheep to determine the pharmacokinetics of orally or i.v. administered arginine and the safety of its long-term supplementation.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Chemicals. HPLC-grade water and methanol were procured from Fisher Scientific. L-Arginine-HCl was obtained from Ajinomoto. All other chemicals were purchased from Sigma. In all experiments, L-arginine-HCl rather than L-arginine base was used to prevent an acid-base imbalance in the infusion solution, drinking water, or milk replacer diet. This study was approved by the Texas A&M University Animal Care and Use Committee.

    General methods. In all experiments, animals were administered a bolus i.v. or oral dose of arginine, followed by the collection of venous blood samples into plain tubes immediately before and at 0.5, 1, 2, 3, 4, and 5 h after administration. The different doses of arginine for sheep, pigs, and rats were chosen because they were shown in preliminary studies to effectively increase arginine concentrations in plasma by 70% at 1-h postinjection. Blood samples were immediately centrifuged at 10,000 x g for 1 min to obtain sera. Serum was deproteinized with an equal volume of 1.5 mol/L HClO₄, neutralized with K₂CO₃, and then diluted 25 times with HPLC water. The diluted samples were stored at –80°C until analysis within 2 wk. Amino acids were determined by HPLC methods involving precolumn derivatization with o-phthaldialdehyde, as previously described (16). Amino acids in samples were quantified on the basis of authentic standards (Sigma Chemicals) using Millenium-32 Software (Waters). Throughout the study, feed intake, health status (regularly examined by attending veterinarians of Texas A&M University), and death (the permanent cessation of all vital bodily functions) of experimental animals were determined as useful endpoints for assessing their tolerance to supplemental arginine, as described for studying amino acids in animal models (17).

    Expt. 1. Arginine pharmacokinetics in nonpregnant and pregnant sheep. Two-year-old nonpregnant and pregnant Suffolk ewes with singleton pregnancy at d 105 of gestation (parity 1) were obtained from the Texas A&M University's Animal Science Teaching, Research and Extension Center and used for the study of arginine pharmacokinetics. Ewes were mated with a fertile Suffolk ram. Before i.v. administration of arginine, the sheep had free access to drinking water and were fed daily 1.6 kg of a corn- and alfalfa-based diet (18) (0.8 kg each at 0700 and 1900) to meet NRC-recommended nutrient requirements (19). On the day of study, 8 h after the morning feeding, individual ewes were administered a single dose i.v. of L-arginine-HCl (equivalent to 27 mg L-arginine/kg body weight) via the jugular vein (n = 5 per group). Blood samples (0.5 mL) were obtained from the contralateral jugular vein. Because arginine is rapidly fermented by microbes in the rumen of ruminants (19), we did not conduct an experiment to determine the pharmacokinetics of orally administered arginine in sheep. To assess the safety of long-term arginine treatment, a catheter was placed into the jugular vein of adult pregnant ewes at d 55 of gestation, as described by Spencer et al. (20). Starting from d 60 of gestation, ewes were administered an i.v. dose of saline or L-arginine-HCl (27 mg arginine/kg body weight every 8 h, equivalent to 81 mg arginine·kg body weight^–1·d^–1). Infusion was terminated at term (d 147 of gestation). There were 10 ewes per treatment group. During the entire period of i.v. infusion of arginine, ewes were fed twice daily 1.6 kg of a corn- and alfalfa-based diet (0.8 kg each at 0700 and 1900) (18). Arginine content in the diet was 0.75% on an as-fed basis and the arginine intake from the basal diet was 150 mg·kg body weight^–1·d^–1.

    Expt. 2. Arginine pharmacokinetics in nonpregnant and pregnant pigs. Pigs were the offspring of Yorkshire x Landrace dams and Duroc x Hampshire sires obtained from the Texas A&M University's Animal Science Teaching, Research and Extension Center. Eleven-month-old nonpregnant pigs and pregnant pigs with multiple fetuses (10–12 per litter) at d 80 of gestation (parity 1) were used for the study of arginine pharmacokinetics. The pigs had free access to drinking water and were fed daily 2 kg of a corn- and soybean meal-based diet (21) (1 kg each at 0700 and 1900) to meet NRC-recommended nutrient requirements (22). On the day of study, 6 h after the morning feeding, individual pigs were administered either i.v. (via the jugular vein) or orally a single dose of L-arginine-HCl (equivalent to 50 mg L-arginine/kg body weight) (n = 6 per group). Blood samples (0.1 mL) were obtained from the ear vein. To assess the safety of long-term arginine treatment, pregnant gilts were fed daily 2 kg of a corn- and soybean meal-based diet (1 kg each at 0700 and 1900) (21) supplemented with 0 or 1.5% L-arginine-HCl (0 or 210 mg arginine·kg body weight^–1·d^–1) between d 30 and 114 (term) of gestation. There were 20 gilts per treatment group. Arginine content in the basal diet was 0.70% on an as-fed basis and the arginine intake from the basal diet was 84 mg·kg body weight^–1·d^–1.

    Expt. 3. Arginine pharmacokinetics in neonatal and young adult pigs. Pigs were the offspring of Yorkshire x Landrace dams and Duroc x Hampshire sires obtained from the Texas A&M University's Animal Science Teaching, Research and Extension Center. Eight-day-old piglets and 6-mo-old pigs (barrows) were used for this study. Neonatal pigs were nursed by sows (23), whereas the young adult pigs had free access to drinking water and were fed daily a corn- and soybean meal-based diet (32 g·kg body weight^–1·d^–1) or 16 g/kg body weight each at 0700 and 1900] to meet NRC-recommended nutrient requirements (22). On the day of study of arginine pharmacokinetics, 2 or 6 h after the morning feeding for neonatal or young adult pigs, individual pigs were administered either i.v. (via the jugular vein) or orally a single dose of L-arginine-HCl (equivalent to 50 mg L-arginine/kg body weight) (n = 6 per group). Blood samples were obtained from the contralateral jugular vein of the neonatal pig (0.5 mL) or the ear vein of the young adult pig (0.1 mL). To assess the safety of long-term arginine treatment, 7-d-old piglets were fed a milk-based diet (10) supplemented with 0 or 0.96% arginine-HCl (0 or 620 mg arginine·kg body weight^–1·d^–1) for 14 d, whereas growing-finishing pigs were fed a corn- and soybean meal-based diet (16) (32 g·kg body weight^–1·d^–1 or 16 g/kg body weight each at 0700 and 1900) supplemented with 0 or 1.21% L-arginine-HCl (equivalent to 320 mg arginine·kg body weight^–1·d^–1) between d 60 and 120 of age. There were 10 neonatal pigs or 20 growing-finishing pigs per treatment group. During the entire period of arginine supplementation, all pigs had free access to feed and drinking water. Arginine contents in the diets for neonatal pigs and growing-finishing pigs were 0.76% (on a dry matter basis) and 1.2% (on an as-fed basis), respectively, and arginine intakes from the basal diets were 530 and 384 mg·kg body weight^–1·d^–1, respectively, for neonatal pigs and growing-finishing pigs.

    Expt. 4. Arginine pharmacokinetics in nondiabetes-prone and diabetic BioBreeding (BB) rats. The BB rat is a well-established animal model of type 1 diabetes mellitus (24). Male nondiabetes-prone BB (BBn) rats and age-matched spontaneously diabetic BB (BBd) rats were purchased from the Health Protection Branch (Ottawa, Canada). They were housed in a temperature- and humidity-controlled facility on a 12-h-light:12-h-dark cycle. BBd rats were administered a daily insulin injection to maintain regular body-weight gain and prevent hyperglycemia (25). All the rats had free access to drinking water and a casein-based diet (26). Male BBn rats and BBd rats (100–105 d of age; 30 d post onset of diabetes) were used for the study of arginine pharmacokinetics. On the day of study, 6 h after the last feeding, individual rats were administered either i.v. (via the upper tail vein) or orally a single dose of L-arginine-HCl (equivalent to 500 mg L-arginine/kg body weight) (n = 6 per group). Blood samples (20 µL) were obtained from the lower tail vein into plain microcentrifuge tubes. To assess the safety of long-term arginine treatment, 100-d-old BBd rats (2 wk post onset of diabetes) were fed a casein-based purified diet (26) and were administered drinking water containing 0 or 1.51% L-arginine-HCl (equivalent to 2.14 g arginine·kg body weight^–1·d^–1) for 10 wk. There were 10 rats per treatment group. During the entire period of arginine supplementation, all rats had free access to feed and drinking water. Arginine content in the basal diet was 0.64% on an as-fed basis and the averaged feed intake was 77 g·kg body weight^–1·d^–1, giving an arginine intake of 493 mg·kg body weight^–1·d^–1 from the basal diet.

    Expt. 5. Arginine pharmacokinetics in lean Zucker rats and obese ZDF rats. The ZDF rat is a well-established animal model of type 2 diabetes mellitus (27). Nine-week-old male lean Zucker rats and obese ZDF rats were obtained from Charles River and fed a Purina 5008 diet. They were housed in a temperature- and humidity-controlled facility on a 12-h-light:12-h-dark cycle. All the rats had free access to drinking water and food. Thirteen-week-old lean Zucker rats and obese ZDF rats (5 wk post onset of diabetes) were used for the study of arginine pharmacokinetics. On the day of study, 6 h after the last feeding, individual rats were administered either i.v. (via the upper tail vein) or orally a single dose of L-arginine-HCl (equivalent to 500 mg L-arginine/kg body weight) (n = 6 per group). Blood samples (20 µL) were obtained from the lower tail vein into plain microcentrifuge tubes. To assess the safety of long-term arginine treatment, 9-wk-old ZDF rats were fed a Purina 5008 diet and were administered drinking water containing 0 or 1.51% L-arginine-HCl (equivalent to 5.70 g arginine·kg body weight^–1·d^–1) for 10 wk. There were 10 rats per treatment group. During the entire period of arginine supplementation, all rats had free access to feed and drinking water. Arginine content in the Purina 5008 diet was 1.44% on an as-fed basis and the arginine intake from the basal diet was 1.50 g·kg body weight^–1·d^–1.

    Data calculation and statistical analysis. Results are expressed as means ± SEM. In the analysis of pharmacokinetics of both i.v. and orally administered arginine, the data were fitted as the increase from baseline values. The model that showed the best fit to the data was chosen for further analysis.

The single exponential model, serum arginine = a x e^(–bxt), was used for analyzing the pharmacokinetics of a bolus i.v. dose of arginine, where a is maximum concentration in serum and b is elimination rate (28). The internal exposure to exogenous arginine was estimated by calculating the area under the concentration-time curve (AUC; a/b), with total clearance (CL) = dose/AUC (29). The maximum concentration of arginine (C_max) in serum was calculated by back-extrapolation of the elimination curve to time zero. The biological half-life (T_1/2; the time taken for serum concentration of arginine to fall by one-half) of exogenous arginine was determined from the elimination curve.

For calculation of the pharmacokinetics of orally dosed arginine, a model with 3 compartments (dosing, serum, and tissue) was utilized: serum arginine = a x [e^(–b1xt)–e^(–b2xt)], where a is a scaling coefficient, with b₁ and b₂ being positive exponents (28). The time at which the C_max in serum was reached is T_max = ln (b₂/b₁)/(b₂–b₁), which upon substitution into the model yields C_max. The internal exposure to oral arginine was estimated by calculating AUC, a x (1/b₁–1/b₂), with CL = (F x dose/AUC_oral) (29). The T_1/2 was calculated from the elimination curve. The absolute bioavailability (F) of orally administered arginine was calculated as AUC_oral/AUC_iv, where AUC_oral and AUC_iv are the AUC for the same oral and i.v. doses of arginine, respectively.

Data were analyzed by ANOVA and the Student-Newman-Keuls multiple comparison test using the SPSS. Probability values <0.05 were considered significant.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Arginine pharmacokinetics in nonpregnant and pregnant ewes. Concentrations of arginine in serum were lower (P < 0.05) in pregnant than in nonpregnant ewes at 0, 0.5, 1, 2, and 3 h after a bolus i.v. dose (Fig. 1). Concentrations of serum arginine returned to the baseline levels at 4 and 5 h after arginine administration in pregnant and nonpregnant ewes, respectively. The AUC, T_1/2, and C_max were 61, 29, and 34% lower (P < 0.01) in pregnant than in nonpregnant ewes, respectively (Table 1). In contrast, the CL was 156% greater (P < 0.01) in pregnant ewes compared with nonpregnant ewes (Table 1).

View larger version (10K): [in this window] [in a new window]   FIGURE 1  Concentrations of arginine in serum of nonpregnant and pregnant ewes after i.v. administration of arginine-HCl (26.7 mg arginine/kg body weight). Data are means ± SEM, n = 5. Results were analyzed by 1-way ANOVA. At the time points of 0, 0.5, 1, 2, and 3 h after a bolus i.v. dose of arginine, concentrations of serum arginine differed (P < 0.05) between the 2 groups of animals. Values at time zero were calculated by back-extrapolation of the elimination curve.

View larger version (17K): [in this window] [in a new window]   FIGURE 2  Concentrations of arginine in serum of nonpregnant and pregnant gilts after i.v. or oral administration of arginine (50 mg arginine/kg body weight). Data are means ± SEM, n = 6. Results were analyzed by 1-way ANOVA. At the time points of 0, 0.5, 1, 2, and 3 h after a bolus i.v. dose of arginine, concentrations of serum arginine differed (P < 0.05) between nonpregnant and pregnant gilts. The values at time zero were calculated by back-extrapolation of the elimination curve. In response to an oral dose of arginine, concentrations of serum arginine differed (P < 0.05) between the 2 groups of gilts at the time points of 0.5, 1, 2, 3, and 4 h.

    Arginine pharmacokinetics in neonatal and young adult pigs. Following i.v. administration of arginine, its concentrations in the serum of 8-d-old pigs were lower (P < 0.05) at 0, 0.5, 1, 2, and 3 h after injection compared with 6-mo-old pigs (Fig. 3A). The AUC, T_1/2, and C_max were 165, 28, and 88% greater (P < 0.05) in young adult pigs than in neonatal pigs, respectively (Table 3). The CL was 62% lower (P < 0.01) in young adult pigs when compared with neonatal pigs (Table 3).

View larger version (17K): [in this window] [in a new window]   FIGURE 3  Concentrations of arginine in serum of neonatal and young-adult pigs after i.v. or oral administration of arginine (50 mg arginine/kg body weight). Data are means ± SEM, n = 6. Results were analyzed by 1-way ANOVA. At the time points of 0, 0.5, 1, 2, and 3 h after a bolus i.v. dose of arginine, concentrations of serum arginine differed (P < 0.05) between 8-d-old and 6-mo-old pigs. The values at time zero were calculated by back-extrapolation of the elimination curve. In response to an oral dose of arginine, concentrations of serum arginine differed (P < 0.05) between the 2 groups of pigs at the time points of 0.5, 1, 2, 3, and 4 h.

    Arginine pharmacokinetics in rats with type-1 diabetes mellitus. Arginine concentrations in serum at 0, 0.5, 1, and 2 h after a bolus i.v. dose were lower (P < 0.05) in BBd than in BBn rats (Fig. 4A). The AUC, T_1/2, and C_max for i.v. administered arginine were 25, 14, and 18% lower (P < 0.05), respectively, in BBd than in BBn rats, whereas the CL was 33% greater (P < 0.05) in BBd rats when compared with BBn rats (Table 4).

View larger version (16K): [in this window] [in a new window]   FIGURE 4  Concentrations of arginine in serum of nondiabetic and BBd rats after i.v. or oral administration of arginine-HCl (500 mg arginine/kg body weight). Data are means ± SEM, n = 6. Results were analyzed by 1-way ANOVA. At the time points of 0, 0.5, 1, and 2 h after a bolus i.v. dose of arginine, concentrations of serum arginine differed (P < 0.05) between BBn and BBd rats. The values at time zero were calculated by back-extrapolation of the elimination curve. In response to an oral dose of arginine, concentrations of serum arginine differed (P < 0.05) between the 2 groups of rats at the time points of 0.5, 1, 2, and 3 h.

    Arginine pharmacokinetics in rats with type-2 diabetes mellitus. Arginine serum concentrations were greater (P < 0.05) in obese ZDF rats than in lean Zucker rats at 0.5, 1, 2, and 3 h after i.v. injection of arginine (Fig. 5A). Serum arginine concentrations at other times did not differ between the 2 groups of animals. The AUC and T_1/2 for a bolus i.v. dose of arginine were 31 and 25% greater (P < 0.05) in obese ZDF rats than in lean Zucker rats, respectively (Table 5). In contrast, the CL was 24% lower (P < 0.05) in obese ZDF rats compared with lean Zucker rats.

View larger version (17K): [in this window] [in a new window]   FIGURE 5  Serum concentrations of arginine in lean Zucker and obese Zucker diabetic rats after i.v. or oral administration of arginine-HCl (500 mg arginine/kg body weight). Data are means ± SEM, n = 6. Results were analyzed by 1-way ANOVA. At the time points of 0, 0.5, 1, 2, and 3 h after a bolus i.v. dose of arginine, concentrations of serum arginine differed (P < 0.05) between Zucker lean and obese ZDF rats. The values at time zero were calculated by back-extrapolation of the elimination curve. In response to an oral dose of arginine, concentrations of serum arginine differed (P < 0.05) between the 2 groups of rats at the time points of 0.5, 1, 2, and 3 h.

    Safety of chronic dietary arginine supplementation to animals. Concentrations of arginine in serum at 0.5, 1, 2, and 3 h after administration increased (P < 0.05) in response to its chronic i.v. administration (for sheep) or dietary supplementation (for pigs and rats) compared with baseline values (data not shown). However, in all the animals studied, concentrations of serum arginine did not differ at 5 h after i.v. or oral administration between arginine-supplemented and nonsupplemented groups. In Expt. 1 and 2, ewes and gilts consumed all the feeds provided daily. In Expt. 3, 4, and 5, feed intake did not differ between arginine-supplemented and nonsupplemented animals (data not shown). Daily i.v. administration of arginine-HCl to adult pregnant ewes (81 mg arginine·kg body weight^–1·d^–1) for 87 d did not result in any undesirable effects of the arginine treatment. Likewise, neonatal pigs, growing-finishing pigs, pregnant gilts, and adult rats tolerated large amounts of chronic supplemental arginine (e.g. 0.62, 0.32, 0.21, and 2.14–5.70 g·kg body weight^–1·d^–1, respectively) administered via enteral diets without the appearance of any adverse effect. In all of our experiments, oral or i.v. administration with arginine did not cause sickness or death in any animal.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Arginine is a major constituent in tissue proteins and is utilized via multiple pathways in animals (3). Arginase is widely expressed in mammalian tissues and is quantitatively the major enzyme for initiating arginine catabolism in animals (4). Accordingly, previous studies with human subjects demonstrate that arginine is cleared rapidly from plasma (7,8). Similarly, our results indicate that all the animals studied rapidly catabolized the supplemental arginine (Figs. 1–5). In response to arginine administration, serum concentrations of ornithine, urea, and proline increased, but those of glutamine and ammonia decreased in a dose-dependent manner in all animals studied (data not shown). Thus, arginine-derived ornithine is rapidly converted into proline via ornithine aminotransferase (OAT) and pyrroline-5-carboxylate reductase (4). In support of this view, high activities of both enzymes occur in tissues of all studied mammals, including pigs, sheep and rats (4). Concentrations of citrulline increased only with very high doses of arginine administration (12), primarily due to increases in its synthesis from ornithine and proline via the intestinal OAT and proline oxidase pathways (30).

Results of this study indicate that, in all the animals receiving i.v. administration of arginine, its elevated concentrations in serum declined and returned to the baseline values within 4–5 h after dosing, with the rates varying with the age and physiological status of the animals (Tables 1–5). The clearance of arginine was higher in pregnant than in nonpregnant animals, in neonatal than in adult animals, in lean than in obese animals, and in type-1 diabetic than in nondiabetic animals (Tables 1–5). As an abundant amino acid in tissue proteins, arginine represents 14% of total nitrogen in the animal body (31). High rates of arginine elimination in neonates are attributed to high rates of protein synthesis and accretion in tissues, particularly skeletal muscle and small intestine (32). Similarly, the rapid growth of the conceptus in pregnant dams, particularly during late gestation (33), is primarily responsible for the high rate of arginine clearance observed in both ewes and pigs (Tables 1 and 2). The clearance rate of arginine was 18% greater in pregnant ewes than in pregnant pigs at the similar relative gestation age (at 70% of gestation) (Tables 1 and 2). This finding may be explained by a high activity of arginase in ovine placentomes (34) and the absence of this enzyme from porcine placenta (35). A high activity of arginase in tissues of type-I diabetic rats (36) is likely responsible for a high rate of arginine clearance in BBd than in BBn rats (Table 4). In contrast, a lower clearance rate of arginine (expressed on a body weight basis) in obese ZDF rats than in lean Zucker rats (Table 5) may be explained by a lower activity of arginase in adipose tissue than in skeletal muscle on a tissue weight basis (G. Wu, unpublished data).

The pharmacokinetics of orally administered arginine is more complex than that of i.v. infused arginine, because the former is regulated not only by intestinal absorption of arginine (7,8) but also by its catabolism in the gastrointestinal tissues (6). Nonetheless, the differences in the clearance of arginine from serum between the studied groups (pregnant vs. nonpregnant; neonatal vs. adult; lean vs. obese; type-1 diabetic vs. nondiabetic) in response to i.v. administration of arginine were generally observed under the experimental conditions of oral administration. With the exception of neonatal pigs (Fig. 3) and obese Zucker rats (Fig. 5), the qualitative differences in the highest serum concentrations of arginine between i.v. and oral administration were noted for all other studied groups. Neonatal pigs lack arginase in the small intestine for arginine degradation (37) and, therefore, nearly all of the enterally delivered arginine that is not utilized for intestinal protein synthesis enters the portal circulation. Consistent with this notion, we found that the absolute bioavailability of oral arginine was 0.92 in 8-d-old pigs, in comparison with the value of 0.63 for young adult pigs (Table 3). In the case of Zucker rats, as noted above, arginine dosing on the basis of the whole body weight may not be optimal for studying its pharmacokinetics, because little is currently known about arginine metabolism in adipose tissue.

Given the rapid clearance of arginine from plasma in animals and humans, it is important to specify the time of blood sampling in studies involving acute or chronic administration of arginine. Even under the conditions of long-term i.v. or oral administration of arginine, we were not able to detect a difference in its serum concentrations between arginine-supplemented and nonsupplemented groups when blood samples were obtained at 5 h after administration. These findings are important because they indicate that: 1) supplemental arginine cannot be stored in serum, and 2) exogenous provision of arginine every 4 h is necessary for achieving a sustained increase in its circulating levels. Interestingly, despite a lack of a sustained increase in plasma concentrations of arginine, dietary supplementation with arginine either twice (9) or once (38) daily to pigs has been reported to improve their reproductive performance. Additionally, i.v. administration of arginine to underfed pregnant ewes every 8 h between d 60 and 114 of gestation had a potential to enhance fetal growth (A. Lassala, unpublished data). Further, i.v. infusion of arginine (20 g over a 2-h period daily for 5 consecutive days) to women with pregnancy-induced hypertension reduced blood pressure (39). These results suggest that an absence of a sustained increase in circulating arginine concentrations under the conditions of daily arginine supplementation is still effective in yielding a beneficial physiological response. It is possible that arginine exerts its nutritional and physiological functions partly through changes in cellular expression of related key genes. In support of this view, arginine regulates expression of inducible NO synthase in activated macrophages (40), GTP cyclohydrolase in endothelial cells (41), and peroxisome-activated proliferator coactivator-1 in adipose tissue (12).

Arginine itself is not toxic (1) and its use as a supplement to diets (usually at <2.5% of dry matter) is generally safe for animals (42,43). However, arginine, lysine, and histidine (basic amino acids) share the same transport systems on the plasma membrane (44), which affects the absorption of arginine into cells. Available evidence shows that excess arginine (generally >2.5% of the diet on a dry matter basis) results in severe adverse effects, including reduced feed intake, impaired growth, and even death, because of an amino acid imbalance (43). Thus, both the nutritional efficacy and the safety of arginine supplementation depend on not only dosage but also dietary contents of other amino acids, particularly basic amino acids. Results of our study indicate that neonatal pigs, growing-finishing pigs, and adult rats can tolerate large amounts of chronic supplemental arginine (e.g. 0.62, 0.32, and 2.14 g·kg body weight^–1·d^–1, respectively) administered via enteral diets, without the appearance of any adverse effect. Further, we demonstrated that either dietary supplementation with arginine-HCl to adult pregnant gilts between d 30 and 114 (term) of gestation at a dose of 210 mg arginine·kg body weight^–1·d^–1 or i.v. administration of arginine-HCl to adult pregnant ewes at a dose of 81 mg arginine·kg body weight^–1·d^–1 between d 60 and 147 (term) of gestation did not result in any undesirable effect of the arginine treatment.

Results from our studies provide a basis for estimating a safe intake of arginine by humans. The sheep is a well-established model for studying placental and fetal development (33), whereas neonatal and adult pigs are similar to humans in most aspects of their digestion, nutrition, metabolism, and physiology (17,45). Thus, based on the i.v. administration of arginine to adult sheep (81 mg·kg body weight^–1·d^–1) a 70-kg adult would be predicted to tolerate a parenteral supplemental dose of at least 6 g arginine/d. Moreover, on the basis of dietary supplementation with arginine at the doses of 0.62, 0.32, and 0.21 g·kg body weight^–1·d^–1 for neonatal, growing-finishing pigs, and adult pigs, respectively, we expect that a 5-kg infant, 30-kg growing child, and 70-kg adult human should tolerate enteral supplemental doses of 3, 10, and 15 g arginine/d, respectively. Further, an adult rat can tolerate oral administration of 2.14–5.70 g arginine·kg body weight^–1·d^–1. Dry matter intake by an adult human is 5.5 g·kg body weight^–1·d^–1 (46), which is 10% of the value for adult rats (11). On the basis of the difference in food intake per kg body weight between rats and humans, a 70-kg adult human will tolerate an enteral supplemental dose of 15–40 g arginine/d. This estimated lower value is the same as that from our study with adult pigs. It is important that arginine be taken in divided doses for the following reasons: 1) to prevent gastrointestinal tract disorders due to abrupt production of large amounts of NO (47); 2) to increase the availability of circulating arginine over a longer period of time (7); and 3) to avoid a potential imbalance among amino acids (48).

Our estimation of safe arginine intake based on animal studies is consistent with the results from human clinical research (49–56). For example, short-term i.v. infusion of arginine at the doses of 0.50, 0.37, and 0.33 g arginine-HCl·kg body weight^–1·d^–1 for infants (50), adults (51), and pregnant women (52), respectively, did not result in any harmful effect. In addition, oral administration of arginine to adults (9 to 15 g/d) for 7 to 12 wk (53,54) had no adverse effect for all the studied subjects. Most adult subjects tolerated higher doses of oral arginine, such as 21 g/d for 4–12 wk (8) or 40 g/d for 7 d (51). However, some human adults experienced occasional side effects of oral arginine at doses higher than 15 or 21 g/d, which included nausea, gastrointestinal discomfort, and diarrhea (8,55) possibly due to a rapid and excess production of NO (47). The median intake of dietary arginine in the U.S. adult population is 4 g/d, with the 90th percentile >7.5 g/d (56). Thus, a 70-kg human subject will normally tolerate chronic parenteral and enteral supplemental doses of 6 and 15 g/d arginine, respectively, in addition to a basal amount of arginine (4–6 g/d) from regular diets.

In summary, animals rapidly catabolize both enterally and parenterally supplemented arginine. The clearance of circulating arginine varies greatly with the age and physiological status of the animals. Neonatal, growing-finishing, and pregnant pigs as well as adult rats can tolerate large amounts of enteral supplemental arginine without the appearance of any adverse effect, as do pregnant ewes receiving i.v. administration of a large dose of arginine-HCl. On the basis of the comparative studies and a consideration of species differences in food intake per kilogram body weight, we estimate that a 70-kg human subject should be able to tolerate long-term parenteral and enteral supplemental doses of 6 and 15 g/d arginine, respectively, in addition to a basal amount of arginine (4–6 g/d) from regular diets.

	ACKNOWLEDGMENTS

 
We thank Mr. Scott Jobgen and Ms. Frances Mutscher for assistance in manuscript preparation, Dr. Sidney Morris and Dr. Virginia Fajt for helpful discussion, as well as Dr. Darrell Knabe, Mr. Kenton Lilie, and Mr. Carey Satterfield for assistance in animal management.

	FOOTNOTES

 
¹ Published in a supplement to The Journal of Nutrition. Presented at the conference "The Sixth Workshop on the Assessment of Adequate and Safe Intake of Dietary Amino Acids" held November 6–7, 2006 in Budapest. The conference was sponsored by the International Council on Amino Acid Science (ICAAS). The organizing committee for the workshop was David H. Baker, Dennis M. Bier, Luc A. Cynober, Yuzo Hayashi, Motoni Kadowaki, Sidney M. Morris, Jr., and Andrew G. Renwick. The Guest Editors for the supplement were: David H. Baker, Dennis M. Bier, Luc A. Cynober, Motoni Kadowaki, Sidney M. Morris, Jr., and Andrew G. Renwick. Disclosures: all Editors and members of the organizing committee received travel support from ICAAS to attend the workshop and an honorarium for organizing the meeting.

² Author disclosures: G. Wu, travel expenses to attend the meeting were paid by ICAAS; F. W. Bazer, T. A. Cudd, W. S. Jobgen, S. W. Kim, A. Lassala, P. Li, J. H. Matis, C. J. Meininger, and T. E. Spencer, no conflicts of interest.

³ Supported in part by grants from the USDA CSREES National Research Initiative Competitive Program (nos. 2001-35203-11247, 2003-35206-13694, 2005-35203-16252, and 2006-00863), from the NIH (nos. 1R21 HD049449 and 5P30ES09106), from the AHA (nos. 9740124N, 0255878Y, and 0655109Y), from the Juvenile Diabetes Research Foundation (no. 1-2002-228), and from the Texas Agricultural Experiment Station (no. H-8200).

⁹ Abbreviations used: AUC, total area under the concentration-time curve; BB, BioBreeding; BBd, diabetic BB; BBn, nondiabetes-prone BB; CL, total clearance; C_max, maximum concentration of arginine; F, absolute bioavailability; NO, nitric oxide; T_1/2, half-life; T_max, time at which maximum concentration occurs; ZDF, Zucker diabetic fatty.

	LITERATURE CITED

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