Convenient method for the determination of arginine and its related compounds in rumen fluid by reversed-phase high-performance liquid chromatography.
In order to clarify arginine (Arg) metabolism by rumen microorganisms and by the tissues of ruminant animals, a convenient method for the simultaneous determination of Arg, citrulline (Cit), ornithine (Orn), proline (Pro) and 5-aminovaleric acid (5AV), and 4-aminobutyric acid (4AB) and lysine (Lys), incidentally, in goat rumen fluid was established by reversed-phase high-performance liquid chromatography (RP-HPLC). The separation was carried out by stepwise isocratic elution with two mobile phases (solvent A and solvent B) on a LiChrospher 100 RP-18 column (150x4.6 mm I.D., 5 microm particle size) equipped with a guard column (4.0x4 mm, 5 microm particle size). Solvent A is composed of acetonitrile-sodium citrate buffer (pH 7.2) (15:85, v/v) containing tetrahydrofuran (5 ml/100 ml), with solvent B comprising acetonitrile-sodium citrate buffer (pH 5.4) (40:60, v/v). Five compounds (Cit, Arg, Pro, 4AB and 5AV) were separated within 33 min in solvent A and the other two (Orn and Lys) in solvent B. Solvent A was automatically switched to solvent B with the help of a valve controller. Complete separation needs 62 min after sample injection in a single chromatogram. Samples were derivatized with 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl) and detected on a fluorescence detector at excitation and emission wavelengths of 263 and 611 nm, respectively. The minimum detectable concentrations (microM) (signal-to-noise ratio, S/N 3:1) of these compounds were: 0.65 for Cit, 0.65 for Arg, 1.9 for Pro, 1.3 for 4AB, 1.9 for 5AV, 0.12 for Orn and 0.48 for Lys. When applied to rumen fluid from goats, recoveries of all compounds added to the rumen fluid were 96.6-100.6% for an intra-day study and 93.9-99.4% for inter-day (5 days) studies. The average contents of Orn, 5AV and Lys in the rumen fluid of three goats before morning feeding were 7.3, 13.5 and 3.6 microM, but Cit, Arg, Pro, and 4AB were not found, although all these four compounds were detected 1 h after feeding. Pro (390 microM) and 5AV (497.6 microM) were highest 1 h after feeding and then decreased. Orn levels before morning feeding were most similar to those after feeding.
Sultana
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Laboratory of Animal Nutrition and Biochemistry, Division of Animal Science, Miyazaki University, Japan. hasu27@hotmail.com
Onodera
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Or-Rashid
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Wadud
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D021542:Amino Acids, Neutral / Q000032:analysis; D000818:Animals; D001120:Arginine / Q000032:analysis* / Q000378:metabolism; D001826:Body Fluids / Q000737:chemistry*; D002021:Buffers; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002956:Citrulline / Q000032:analysis; D005766:Gastrointestinal Contents / Q000737:chemistry*; D006041:Goats; D006863:Hydrogen-Ion Concentration; D009952:Ornithine / Q000032:analysis; D011392:Proline / Q000032:analysis; D015203:Reproducibility of Results; D012417:Rumen / Q000378:metabolism* / Q000382:microbiology; D013997:Time Factors
D021542:Amino Acids, Neutral; D002021:Buffers; D002956:Citrulline; C013809:5-aminovaleric acid; D001120:Arginine; D011392:Proline; D009952:Ornithine