Metabolic studies reveal that ruminal microbes of adult steers do not degrade rumen-protected or unprotected L-citrulline.

In vitro and in vivo experiments were conducted to determine the metabolism of rumen-protected or unprotected l-citrulline (Cit) plus l-glutamine (Gln) by ruminal microbes. In the in vitro experiment, whole ruminal fluid (3 mL, containing microorganisms) from steers was incubated at 37 ºC with 5 mM Cit plus 6 mM Gln (in a rumen-protected or unprotected form) for 0, 0.5, 2, or 4 h after which times 50 µL samples were collected for AA and ammonia analyses. In the in vivo experiment, at 0.5 h before and 0, 0.5, 1, 2, 4, and 6 h after cannulated adult steers consumed 0.56 kg dried-distillers' grain mixed with 70 g Cit plus 70 g Gln (in a rumen-protected or unprotected form), samples of ruminal fluid and jugular venous blood were obtained for AA analyses. Results from both in vitro and in vivo experiments demonstrated extensive hydrolysis of rumen-unprotected Gln into glutamate, but little degradation of the rumen-protected Gln or rumen-protected and unprotected Cit by ruminal microbes. Concentrations of Cit and arginine in the plasma of steers consuming rumen-protected or unprotected AA increased at 1 and 2 h after the meal, respectively, when compared with values at 0 h. Collectively, these novel findings indicate that ruminal microbes of adult steers do not degrade extracellular Cit in a rumen-protected or unprotected form. Our results refute the view that all dietary AAs are extensively catabolized by ruminal microorganisms and also have important implications for dietary supplementation with Cit to ruminants to enhance the concentration of arginine in their plasma and their productivity.

Ruminal microbes of adult steers do not degrade extracellular L-citrulline and have a limited ability to metabolize extracellular L-glutamate1,2.

The microbial population within the rumen has long been considered to have the capability of extensively degrading all dietary AA. Results from our feeding trials revealed that this dogma is not correct. In vitro studies were conducted to test the hypothesis that certain AA undergo little degradation by ruminal microbes. Whole ruminal fluid (3 mL, containing microorganisms) from cannulated adult steers (~500 kg, n = 6) was incubated at 37 °C with 5 mM l-glutamine, l-glutamate, l-arginine, or l-citrulline for 0, 0.5, 1, and 2 h to determine time-dependent changes in the metabolism of these AA. Additional ruminal fluid was incubated with 0, 0.5, 2 or 5 mM l-glutamine, l-glutamate, l-arginine, or l-citrulline for 2 h to determine dose-dependent changes in their metabolism. An aliquot (50 µL) of the incubation solution was collected at the predetermined time points for AA analyses. There was extensive hydrolysis of l-glutamine into l-glutamate and ammonia, and l-arginine into l-ornithine, l-proline, and ammonia, but the near absence of catabolism of extracellular l-glutamate and no degradation of extracellular l-citrulline by ruminal microbes. There was little uptake of 14C-labeled l-glutamate and no detectable uptake of 14C-labeled l-citrulline by the cells. These results indicate, for the first time, that ruminal microbes of adult steers do not degrade extracellular l-citrulline and that metabolism of extracellular l-glutamate is negligible compared with their ability to extensively catabolize extracellular l-arginine and l-glutamine.

Decreased hippocampal homoarginine and increased nitric oxide and nitric oxide synthase levels in rats parallel training in a radial arm maze.

L-homoarginine (hArg) is derived from enzymatic guanidination of lysine. It was demonstrated that hArg is a substrate for nitric oxide (NO) synthesis, blocks lysine transport and inhibits the uptake of arginine into synaptosomes and modulates GABA responses ex vivo. As there is limited information on its physiological roles in the brain, the aim of the study was to show whether hippocampal or frontal lobe (FL) hArg is paralleling training in the radial arm maze (RAM) or NO formation. Hippocampi and FL of male Sprague-Dawley rats were taken from trained or yoked in a RAM. Then hArg and metabolites, NO and NO synthase (NOS) were determined by standard methods. The animals learned the task in the RAM showing significant reduction of working memory errors. hArg showed decreased levels in both brain regions of trained animals as compared to yoked animals. Nitrate plus nitrite (NOx) concentrations and NOS activity were significantly increased in hippocampi, F(1,36) = 170.5; P ≤ 0.0001 and FL, F(1,36) = 74.67; P ≤ 0.0001 of trained animals as compared to yoked animals. Levels of hArg were negatively correlated with NOx in hippocampus (r = -0.6355; P = 0.0483) but not in FL and with lysine in the FL (r = -0.6650; P = 0.0358). NOx levels were positively correlated with NOS in both the hippocampus (r = 0.7474; P = 0.0129) and FL (r = 0.9563; P ≤  0.0001). These novel findings indicate that hArg is linked to NO formation in hippocampus but not in FL and is paralleling spatial memory in the RAM.

Whole-body synthesis of L-homoarginine in pigs and rats supplemented with L-arginine.

Recent studies suggest an important role for L-homoarginine in cardiovascular, hepatic and neurological functions, as well as the regulation of glucose metabolism. However, little is known about whole-body L-homoarginine synthesis or its response to dietary L-arginine intake in animals. Four series of experiments were conducted to determine L-homoarginine synthesis and catabolism in pigs and rats. In Experiment 1, male and female pigs were fed a corn- and soybean meal-based diet supplemented with 0.0-2.42 % L-arginine-HCl. In Experiment 2, male and female rats were fed a casein-based diet, while receiving drinking water containing supplemental L-arginine-HCl to provide 0.0-3.6 g L-arginine/kg body-weight/day. In both experiments, urine collected from the animals for 24 h was analyzed for L-homoarginine and related metabolites. In Experiment 3, pigs and rats received a single oral dose of 1 or 10 mg L-homoarginine/kg body-weight, respectively, and their urine was collected for 24 h for analyses of L-homoarginine and related substances. In Experiment 4, slices of pig and rat tissues (including liver, brain, kidney, heart, and skeletal-muscle) were incubated for 1 h in Krebs-bicarbonate buffer containing 5 or 50 µM L-homoarginine. Our results indicated that: (a) animal tissues did not degrade L-homoarginine in the presence of physiological concentrations of other amino-acids; (b) 95-96 % of orally administered L-homoarginine was recovered in urine; (c) L-homoarginine was quantitatively a minor product of L-arginineg catabolism in the body; and (d) dietary L-arginine supplementation dose-dependently increased whole-body L-homoarginine synthesis. These novel findings provide a new framework for future studies of L-homoarginine metabolism and physiology in animals and humans.

Analysis of L-homoarginine in biological samples by HPLC involving precolumn derivatization with o-phthalaldehyde and N-acetyl-L-cysteine.

L-Homoarginine (hArg) may play a role in regulating the metabolism of its structural homologue L-arginine via multiple pathways (including nitric oxide synthase) in animals. Accurate measurement of hArg is essential for studying its synthesis and utilization by cells and the whole body. Here, we describe a simple, sensitive and automated method for analysis of hArg in biological samples by high-performance liquid chromatography involving precolumn derivatization with o-phthalaldehyde (OPA) and N-acetyl-L-cysteine (NAC) as the thiol. The hArg-OPA-NAC derivative was separated at 25 °C on a reversed-phase C18 material and detected by fluorescence at excitation and emission wavelengths of 340 and 450 nm, respectively. The total running time for one sample (including the time for column regeneration) was 20 min, with the retention time for hArg being 10.03 min. The limit of detection was 188 fmol hArg, which was equivalent to 12 nM hArg in the 160-µl assay mixture. The assay was linear between 1.0 and 80 pmol hArg injected into the HPLC column (equivalent to 0.0625 and 5 µM hArg in the 160-µl assay mixture, respectively). The precision (relative deviation, %) and bias (relative error, %) of the HPLC method were 0.52-1.16 and 0.42-1.12, respectively, for aqueous solutions of hArg and for various biological samples (e.g., plasma, liver, brain and kidney). This is a highly sensitive, accurate, efficient and easily automated method for analysis of hArg in biological samples and provides a useful tool for studying the biochemistry, nutrition, physiology and pharmacology of hArg and arginine in animals and humans.