Introduction

l-Homoarginine (hArg), a structural homologue of l-arginine (Arg), was discovered as a naturally occurring substance in plants (Bell 1962). Subsequently, synthesis of hArg from Arg and l-lysine in animals and humans was reported (Ryan and Wells 1964; Ryan et al. 1968, 1969). It is now known that rat liver, kidneys and the small intestine (Frenay et al. 2015; Hou et al. 2015; Pilz et al. 2015a), as well as human brain (Bernstein et al. 2015) possess the capacity for hArg synthesis. The responsible enzyme remains unknown (Tsikas and Kayacelebi 2014) and may be arginine:glycine amidinotransferase (AGAT) (Davids et al. 2012; Ryan et al. 1969).

Although recent studies suggest an important role for hArg in cardiovascular (Atzler et al. 2015; Pilz et al. 2015b), hepatic (Pilz et al. 2015a) and neurological (Bernstein et al. 2015) functions, as well as the regulation of glucose metabolism (Stockebrand et al. 2015), little is known about whole-body hArg synthesis or its responses to dietary Arg intake in animals (Tsikas and Wu 2015). The objective of this study was to determine hArg production in two animal models (pigs and rats) supplemented with various amounts of dietary Arg. Our results provide, for the first time, quantitative data on Arg metabolism via the hArg pathway and effects of Arg consumption on whole-body hArg synthesis in mammals.

Materials and methods

l-Arginine-HCl and l-alanine were products of Ajinomoto Inc. (Tokyo, Japan). Their purity was >99.9 %, as analyzed by high-performance liquid chromatography (HPLC) (Wu and Meininger 2008). hArg was purchased from Sigma Chemicals (St. Louis, MO, USA). The sources of other chemicals were the same as described previously (Dai et al. 2014b; Hou et al. 2015; Hu et al. 2015). The diets and drinking water for pigs and rats contained no hArg, nitrite, nitrate, creatine, creatinine, agmatine, or methylarginines. The experimental procedures were approved by the Institutional Agricultural Animal Care and Use Committee of Texas A&M University.

Experiment 1: effects of dietary Arg supplementation on urinary excretion of hArg in pigs

Pigs used for this study were the offspring of Yorkshire × Landrace dams and Duroc × Hampshire sires, and were maintained at the Texas A&M University Swine Center. Piglets were weaned at 21 days of age to a conventional corn- and soybean meal-based diet containing 21.0 % crude protein and 1.35 % Arg, as described previously (Hu et al. 2015). At 30 days of age, 18 male or 18 female pigs with similar body weights were assigned randomly to one of three treatment groups (0, 1.21, and 2.42 % Arg-HCl) on the basis of litter origin. There were 6 males and 6 females per dietary group. The three diets were prepared by adding 0, 1.21, or 2.42 % Arg-HCl to the basal diet at the expense of cornstarch. These doses of Arg-HCl provided 0, 1, and 2 % Arg, respectively. Isonitrogenous amounts of l-alanine were added to the diets supplemented with 0 and 1.21 % Arg-HCl (Hu et al. 2015). The animals were housed individually in pens at 23–25 °C and 55–60 % relative humidity. During the entire experimental period, pigs had free access to drinking water and were offered their respective diets at 31.5 g feed per kg body weight per day (divided in two equal meals at 8:00 AM and 5:00 PM). The pigs consumed all the feed provided each day. In the last week of the study, pigs were placed individually in metabolism cages, continuously fed their respective diets, and had free access to drinking water (double-distilled and deionized water). On day 120 of age, a 24-h urine collection into a bottle (containing antibiotics) was made from each pig (Wu et al. 1999). After the completion of urine collection, jugular vein blood samples (2 ml) were collected, into EDTA-containing tubes, from each pig at 4 h after feeding. The blood samples were centrifuged immediately at 10,000 g for 1 min. The supernatant fluid (plasma) was collected and then stored at −80 °C until analyzed.

Experiment 2: effects of dietary Arg supplementation on urinary excretion of hArg in rats

Male and female Sprague–Dawley rats (5 weeks of age) were purchased from Harlan Laboratories (Indianapolis, IN). Upon arrival at the Texas A&M University Kleberg animal facilities, all rats were housed individually in carbonate cages in an animal room at 25 °C and 60 % relative humidity with a 12-h light:12-h dark cycle. During a 1-week period of adaptation, the animals were fed a regular rodent diet (Product Cat #8604, Harlan Teklad) and had free access to drinking water (double-distilled and deionized water). At 6 weeks of age, rats were assigned randomly to one of three treatment groups (0, 2.18 or 4.35 g Arg-HCl/kg body weight/day). These doses of Arg-HCl provided 0, 1.8 or 3.6 g Arg/kg body weight/day, respectively. Isonitrogenous amounts of l-alanine were added to the drinking water (Yang et al. 2015). There were 6 male and 6 female rats per dietary group. During the entire period of the experiment, rats were fed a semi-purified diet containing 4.3 % fat (Jobgen et al. 2009) and had free access to their respective drinking water (double-distilled and deionized water). This enteral diet contained 0.61 % Arg and 0.44 % l-alanine, as analyzed by HPLC (Dai et al. 2014a). Because oral arginine administration of Arg to rats via drinking water tended to increase food intake (Jobgen et al. 2009), Arg-supplemented rats were individually pair-fed with rats in the isonitrogenous control group on a per kg body weight basis to ensure similar intakes of all nutrients (except for arginine and alanine) among the three groups. Consumption of food and water by rats was determined daily. Concentrations of Arg-HCl in drinking water were adjusted daily according to the volume of water consumed by rats to provide intakes of 0, 2.18 or 4.35 g Arg-HCl/kg body weight/day, as indicated previously. In the last week of the study, rats were placed individually in metabolism cages, continuously fed their respective diets, and had free access to drinking water (double-distilled and deionized water). On day 132 of age, 24-h urine collection into a bottle (containing antibiotics) was made from each rat (Wu et al. 1999). After the completion of urine collection, rats were food deprived for 5 h. Thereafter, rats were anesthetized with CO2 and euthanized by cervical dislocation. Cardiac blood samples (2 ml) were collected into heparinized tubes and centrifuged immediately at 10,000 g for 1 min. The supernatant fluid (plasma) was collected and then stored at −80 °C until analyzed.

Experiment 3: recovery of orally administered hArg in the urine of pigs and rats

Three-month-old male (n = 6) and female (n = 6) pigs (44 and 40 kg, respectively) and 3-month-old male (n = 6) and female (n = 6) rats (365 and 240 g, respectively) were placed individually in their metabolism cages and fed their respective diets, as described above. Pigs and rats received a single oral dose of hArg-HCl (dissolved in water) at 1 or 10 mg hArg/kg body weight, respectively. The volume of water was 5 and 1 ml for pigs and rats, respectively. Urine was then collected for 24 h, and then blood samples were obtained from the animals, as described previously. The amount of hArg in the 24-h urine excreted before the day of oral hArg administration was used to correct for basal amounts of hArg excreted into urine and thus calculate the recovery of orally administered hArg in the urine of animals.

Experiment 4: catabolism of hArg by animal tissues

Pigs and rats were fed as described in Exp. 1 and Exp. 2. Male (n = 6) and female (n = 6) pigs (44 and 40 kg, respectively; 121 days of age), as well as 133-day-old male (n = 6) and female (n = 6) rats (365 and 240 g, respectively) were euthanized, as we described previously (Hu et al. 2015; Yang et al. 2015). One hundred milligram slices of tissues (small intestine, large intestine liver, pancreas, spleen, heart, brain, kidney, skeletal muscle, white adipose tissue, thymus, mesenteric lymph nodes, skin, testes, or ovaries) were incubated at 37 °C for 1 h in the presence of 2 ml oxygenated Krebs bicarbonate buffer (95 % O2/5 % CO2, pH 7.4; Wu et al. 1994) containing 5 mM d-glucose, 5 or 50 µM hArg, and physiological concentrations of other AA (except for lysine) (Jobgen et al. 2009; Wu et al. 1997). Lysine was not included in the incubation medium to prevent hArg synthesis (Yang et al. 2015). At the end of the 0 and 1-h incubation period, 0.1 ml of 1.5 M HClO4 was added to the incubation medium. The acidified medium was neutralized with 0.05 ml of 2 M K2CO3. The whole tissue extract was analyzed for hArg.

Chemical analyses

Plasma and urine samples (1 ml) were deproteinized with an equal volume of 1.5 M HClO4, followed by addition of 0.5 ml 2 M K2CO3. The neutralized extracts were used for chemical analyses. Specifically, hArg, agmatine and Arg were determined by HPLC methods involving precolumn derivatization with o-phthaldialdehyde as described previously (Dai et al. 2014b; Hou et al. 2015; Wu and Meininger 2008). Creatinine was determined using a kit from Sigma Chemicals (St. Louis, MO, USA). NOx (nitrite plus nitrate) was measured by an HPLC method involving the enzymatic conversion of nitrate into nitrite and the subsequent derivatization of nitrite with 2,3-diaminonaphthalene to form 2,3-naphthotriazole (Jobgen et al. 2007). For determination of asymmetric dimethylarginine, NG-monomethyl arginine, and symmetric dimethylarginine, sample cleanup was performed using the Oasis MCX SPE column (1 ml, 30 mg) before HPLC analysis (Wu and Meininger 2008).

Statistical analysis

Results are expressed as mean ± SEM. Statistical analyses of data were performed by one-way analysis of variance using the General Linear Models procedures (Assaad et al. 2014). Differences among treatment means were determined using the Student–Newman–Keuls multiple comparison method (Assaad et al. 2014). A probability value of ≤0.05 was taken to indicate statistical significance.

Results

Concentrations of hArg and related metabolites in the plasma and urine of pigs and rats

Data on concentrations of hArg and related metabolites in the plasma and urine of pigs are summarized in Table 1. Male and female pigs responded similarly to dietary Arg supplementation. Specifically, concentrations of Arg, hArg and NOx in plasma, as well as concentrations of hArg, creatinine and NOx increased (P < 0.05) in a dose-dependent manner with dietary supplementation of 0, 315, and 630 mg Arg/kg body weight per day. Interestingly, concentrations of creatinine in plasma or concentrations of Arg in urine did not differ among treatment groups within each sex. Concentrations of methylarginines (formed by the enzymatic methylation of Arg residues in proteins and released by proteolysis) in the plasma or urine of pigs were not affected by dietary Arg supplementation (Table 2). Similar results were observed for rats supplemented with 0, 1.8 and 3.6 g Arg/kg body weight per day (Tables 3, 4).

Table 1 Concentrations of metabolites in the plasma and urine of pigs supplemented with or without l-arginine for 13 weeks
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Table 2 Concentrations of methylarginines in the plasma and urine of pigs supplemented with or without l-arginine for 13 weeks
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Table 3 Concentrations of metabolites in the plasma and urine of rats supplemented with or without l-arginine for 13 weeks
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Table 4 Concentrations of methylarginines in the plasma and urine of rats supplemented with or without l-arginine for 13 weeks
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In both pigs and rats, concentrations of hArg in plasma and urine were relatively low, compared with Arg, creatinine and NOx (Tables 1, 3). With reabsorption of water by kidney tubules, hArg was concentrated in the urine. While concentrations of creatinine and NOx in plasma were lower than those of Arg, concentrations of creatinine and NOx in urine were about 700 and 10–20 times those of Arg, respectively. Thus, reabsorption of creatinine and NOx by kidney tubules was limited in both pigs and rats.

Urinary excretion of hArg and related metabolites in pigs and rats

The volume of urine excreted by male and female pigs was not affected by dietary supplementation with 0, 315 and 630 mg Arg/kg body weight per day (Table 5). Likewise, urinary excretion of Arg did not differ among these three groups of pigs. In contrast, dietary supplementation with Arg dose-dependently increased (P < 0.05) urinary excretion of hArg and NOx in male and female pigs. When data from male and female pigs were combined, diertary supplementation with 0, 315 and 630 mg Arg/kg body weight/day enhanced (P < 0.05) urinary excretion of creatinine and agmatine in a dose-dependent manner (Table 6). Similar results were obtained for rats supplemented with 0, 1.8 and 3.6 g Arg/kg body weight per day (Table 7). In both pigs and rats, urinary excretion of hArg and agmatine was much lower than that of Arg, creatinine and NOx.

Table 5 Urinary excretion of metabolites in pigs supplemented with or without l-arginine for 13 weeks
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Table 6 Concentrations of creatinine and agmatine in plasma, as well as urinary excretion in pigs and rats supplemented with or without l-arginine for 13 weeks
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Table 7 Urinary excretion of metabolites in rats supplemented with or without l-arginine for 13 weeks
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Recovery of orally administered hArg in the urine of pigs and rats

At 24 h after oral administration of hArg, concentrations of hArg in the plasma of pigs and rats (Table 8) were similar to those for animals not receiving an oral dose of hArg (Tables 1, 2). Approximately 96 and 95 % of orally administered hArg was recovered in the urine of pigs and rats, respectively (Table 8). The urinary recovery of hArg did not differ between males and females in either animal species. In both pigs and rats, oral administration of hArg did not affect (P > 0.05) concentrations of Arg, creatinine, agmatine, methylarginines, or NOx in plasma and urine, or urinary excretion of these metabolites.

Table 8 Recovery of orally administered hArg in the urine of pigs and rats
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Degradation of hArg by animal tissues

The limit of detection (defined as a signal-to-noise ratio of 3:1) for our determination of hArg in tissue extracts was 38.4 pmol/ml (Hou et al. 2015). Based on the HPLC analysis of hArg in neutralized tissue extracts (medium plus tissue) before and after a 1-h incubation period, there was no detectable disappearance of hArg from the incubation medium of any animal tissue studied, which included the small intestine, large intestine liver, pancreas, spleen, heart, brain, kidney, skeletal muscle, white adipose tissue, thymus, mesenteric lymph nodes, skin, testes, and ovaries from male and female pigs as well as male and female rats. These results indicate that pig and rat tissues do not degrade hArg in the presence of physiological concentrations of other AA.

Discussion

Arg is a substrate for the synthesis of proteins, hArg, NO, ornithine (the precursor of polyamines, proline, and glutamate), creatine, and agmatine in mammals (Brosnan and Brosnan 2007; Morris 2007; Tsikas and Wu 2015; Wu and Morris 1998). Although concentrations of hArg in tissues and physiological fluids (e.g., plasma, brain, and urine) have been reported for humans (Carmann et al. 2015; Haghikia et al. 2015; Hörster et al. 2015; May et al. 2015), little is known about hArg production in the whole body. Similarly, there is a paucity of information about effects of dietary Arg intake on hArg synthesis in animals or humans. Much-needed data on hArg synthesis and catabolism will expand our knowledge about quantitative aspects of Arg metabolism in the body.

hArg is a chemically stable metabolite in both humans and rats (Kayacelebi et al. 2015a, b; Yang et al. 2015). The major site of hArg synthesis may be the liver and kidneys (Ryan et al. 1969). hArg is not degraded by arginase and other enzymes in animal tissues (the present study), and is not likely a significant source of NO when present at physiologically low concentrations in plasma (~2 µM), as compared with 100–200 µM Arg (Wu et al. 2009). Thus, in vivo catabolism of hArg is limited in animals. Furthermore, enterocytes can absorb dietary hArg from the lumen of the small intestine via cationic AA transporters (Monné et al. 2015), but there is little uptake of hArg from arterial blood into the small intestine (Rutherfurd 2015; Yin et al. 2015). Therefore, urinary excretion of hArg can be a useful indicator of its whole-body production. This view is supported by our findings that 96 and 95 % of orally administered hArg were recovered in the urine of pigs and rats, respectively (Table 8). The unrecovered hArg may be lost through the skin and feces, or utilized by bacteria in the small intestine. In post-weaning pigs and rats, dietary Arg is extensively degraded by the gastrointestinal tract as 40 % of orally administered Arg does not enter the portal circulation (Wu 1998).

Intake of protein-bound Arg by pigs was 425 mg/kg body weight/day. With a 90 % digestibility, Arg derived from dietary protein was 382 mg/kg body weight/day. In pigs supplemented with 0, 315 and 630 mg Arg/kg body weight/day, total Arg intake was 382, 697, and 1012 mg/kg body weight/day. Urinary excretion of hArg represented <0.025 % of Arg intake in pigs. Likewise, the intake of protein-bound Arg by male and female rats was 278 and 302 mg/kg body weight/day, respectively. With a 90 % digestibility, Arg derived from the dietary protein was 264 and 287 mg/kg body weight/day in male and female rats, respectively. In rats supplemented with 0, 1.8 and 3.6 g Arg/kg body weight/day, total Arg intake by male rats was 0.26, 2.06, and 3.86 g/kg body weight/day, and total Arg intake by female rats was 0.29, 2.09, and 3.89 g/kg body weight/day. Urinary excretion of hArg represented <0.045 % of Arg intake in rats. Thus, hArg was quantitatively a minor metabolite of Arg in both pigs and rats. This conclusion, however, should not be taken to indicate a minor role for hArg in animal physiology and pathophysiology (Kayacelebi et al. 2015a, b, c; Khalil et al. 2015; Langen et al. 2015; Papageorgiou et al. 2015; Sasso et al. 2015). Future studies are warranted to determine effects of hArg in nutrient absorption, blood circulation, pregnancy, lactation, and metabolic control.

A novel and important finding from the present study is that increasing dietary intake of Arg dose-dependently enhanced whole-body hArg synthesis and concentrations of hArg in plasma in both pigs and rats (Tables 1, 2). Thus, even at high intakes of Arg (e.g., up to 630 and 3600 mg/kg body weight/day, respectively, in pigs and rats), the enzyme for hArg production did not appear to be saturated in animals and renal AGAT activity was further increased (Yang et al. 2015). This provides a basis for oral administration of Arg to increase hArg availability in animals and humans. Likewise, urinary output of hArg is markedly increased in patients with hyperargininemia due to arginase I deficiency (Schlune et al. 2015). Therefore, we suggest that low extracellular concentrations of Arg can limit hArg synthesis in vivo.

As noted previously, hArg is a poor substrate of NO synthase. At high concentrations (e.g., 1 mM), hArg can even inhibit NOS (Moali 1998; Schini et al. 1991). The Km value of NOS for hArg is about 20 times greater than that for Arg (Moali 1998). Given an exceedingly low concentrations of hArg (~2 µM) in plasma (Tables 1, 2) relative to those of Arg (100–250 µM) (Wu et al. 2009), it is unlikely that hArg is a significant substrate for whole-body NO production under physiological conditions. While rat liver arginase may use hArg as a substrate, its K m value for hArg is 7.2 mM (Reczkowski and Ash 1994), which is about 70 times that for concentrations of hArg in the liver (Yang et al. 2015). The V max values of rat liver arginase for Arg and hArg are 4380 and 34 µmol/min/mg protein, respectively (Reczkowski and Ash 1994). This indicates an exceedingly low activity of purified arginase toward hArg as compared with Arg. Thus, catabolism of hArg by arginase is likely negligible in mammals. In support of this notion, results of our research indicate that a number of tissues from pigs and rats do not degrade hArg in the presence of physiological concentrations of other AA found in the plasma of animals (the current study). These tissues include the small intestine, large intestine liver, pancreas, spleen, heart, brain, kidney, skeletal muscle, white adipose tissue, thymus, mesenteric lymph nodes, skin, testes, and ovaries. Therefore, we suggest that virtually little hArg is catabolized in animals and endogenously synthesized hArg is quantitatively excreted in their urine.

In conclusion, hArg was quantitatively a minor product of Arg metabolism in pigs and rats. Approximately 95–96 % of orally administered hArg was recovered in urine, indicating limited catabolism of hArg in the body. Animal tissues do not degrade hArg in the presence of physiological concentrations of other AA, and thus its urinary excretion is a valid indicator of in vivo production of hArg. Dietary supplementation with Arg increased whole-body hArg synthesis in a dose-dependent manner. These novel results provide a new framework for future studies of hArg metabolism, as well as the roles for hArg in animal and human physiology.