Methods of Finding Arginine in Urine, Food, and Plasma Using HPLC
Amino acids are organic compounds having both carboxyl and amino groups - Methods of Finding Arginine in Urine, Food, and Plasma Using HPLC introduction. Arginine, one of the twenty nutritionally important amino acids, can be made by the body. It has some significant functions in the body such as assisting in wound healing, ammonia removal, stimulating immune function, cell division, hormone secretion, and production of nitric oxide in our body (Healthnotes, 2004). Arginine is an essential amino acid needed by juvenile mammals for their optimum growth. Not only is it the most abundant nitrogen carrier in proteinaceous tissues. It is also used by multiple pathways. These pathways include arginase, nitric oxide (NO)4 synthase, arginine:glycine amidinotransferase, and arginyl-tRNA synthetase. It also serves an important role for synthesis of creatine, proline, glutamate, polyamines, and NO, arginine (Guoyao, Knabe, & Sung, 2004). One method that could help in testing for arginine in substances like urine, food, and plasma is high-performance liquid chromatography or HPLC.
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For finding arginine in urine, first, the urine samples were randomly collected from the subjects. The urine samples were then frozen at 220 °C. Within two weeks, the urine samples were assayed. Based on Venta’s (2001) study entitled “Year-Long Validation Study and Reference Values for Urinary Amino Acids Using a Reversed-Phase HPLC Method”, arginine concentrations in urine can be measured using RP-HPLC using the Pico-Tag method. The system has a Hewlett-Packard Vectra 486 computer that would run the Baseline 810 workstation; there are two Model 510 pumps, an autosampler of Model 712 WISP, a temperature control module, set at 46 °C, and an absorbance detector of Model 484, set at 254 nm. All the chemicals and solvents used in the study were bought from Sigma-Aldrich and Merck. A MilliQ water purification system from Millipore was used for generating HPLC-grade water. For the analysis, the samples were first thawed at room temperature before subjecting them for 15 min. of centrifugation at 3000g. Then, through solid phase extraction the clear supernatants were further cleaned. Next, 1 mL of each sample diluted 1:1 with 400 mmol/L methionine sulfone in 0.1 mol/L HCl was applied to a Sep-Pack Plus C18 cartridge preconditioned at 10 mL of methanol, 10 mL of water on a Sep-Pack vacuum manifold (Waters). The purified amino acids were obtained by collecting the initial pass-through sample with two eluates: first, an eluate of 1.5 mL of 0.1 mol/L HCl, then of 2.5 mL of 300 mL/L acetonitrile in 0.1 mol/L HCl. Derivatization then followed which consists of three evaporation steps in a Waters vacuum station. The first evaporation step involved drying on the calibration mixture (25mL) and urine eluates (50 mL). The second step involved treatment with 10 mL of methanol-1mol/L sodium acetate-triethylamine (2:2:1 by volume) before drying. And the third step involved treatment for 10 min with 20 mLof derivatization reagent [methanol-triethylamine-water-PITC (Pierce), 7:1:1:1 by volume] before drying. After derivatization, the C18 Pico-Tag physiological free amino acid column [300 3 3.9 mm (i.d.)] with a binary gradient of Waters eluents 1 and 2 at a flow rate of 1.0 mL/min was used to separate the derivatives of amino acids. Using the kinetic Jaffe reaction in a Hitachi 911 analyzer, the urinary amino acid was measured the (Venta, 2001). For calibration and quality control, the derivatized calibrators, controls, and urine samples were dissolved in 100 mL of the sample diluent. 10 mL of these solutions was injected. Every two months, two commercial solutions of acidneutral and basic amino acids in 0.1 mol/L HCl was used to prepare an derivatize a calibration mixture. Other solutions such as glutamine and argininosuccinic acid (Sigma-Aldrich) in water, stored at 220 °C was also used. The final mixture has 400 mmol/L of each amino acid except for cystine which is 200 mmol/L. Based on the reference peaks for retention time, the amino acid derivatives were identified. To calculate the amino acid concentrations, the ratio of the peak area of each amino acid to the internal standard was used. Using the Lyphochek Quantitative Urine Control2 (Bio-Rad), the within- and between-run precision was continuously checked. Two urine pools were prepared for the recovery study. Five samples of each pool were enriched with a concentrate of the amino acid calibration mixture (200 mmol/L). Finally, statistical analysis followed (Venta, 2001).
Arginine concentrations in plasma can be measured using a method cited by Cakir et al. in a study entitled “Elevated plasma concentration of asymmetric dimethylarginine that is reduced by single dose testosterone administration in idiopathic hypogonadotropic hypogonadism patients” (2005). First, fasting blood samples from patients and controls were collected after overnight fasting. The blood samples were then put on ice. Next, the venous blood samples centrifuged within less than 60 min were store at -80°C until the L-arginine levels can be measured. 20 mg 5-sulfosalisilic acid was added to 1 ml serum. And after leaving the mixture in an ice bath for 10 min, the L-arginine aws measured. At 2000 x g for 10 min of centrifugation, the precipitated protein was removed. Ten microliters of supernatant that was filtered through a 0.2-µm pore size filter was mixed with 100 µl derivatization reagent. The derivatization reagent was prepared by dissolving 10 mg o-phtaldialdehyde in 0.5 ml methanol, 2 ml 0.4 M borate buffer (pH 10.0), with 30 µl 2-mercaptoethanol. The mixture was then injected into the chromatographic system. Using a 150 x 4-mm interior diameter Nova-Pak C18 column with a particle size of 5 µm, L-arginine was separated.A mobile phase of 50 mM sodium acetate (pH 6.8), methanol, and tetrahydrofurane was used at a flow rate of 1.0 ml/min. Finally, for quantification, the areas of peaks identified by fluorescent detector were used (Cakir, Ozcan, Yaman, Akgul, Bilgi, Erbil, et al., 2005).
Arginine in food can be detected based on the method performed Linget et al in a study entitled “On-line dialysis with HPLC for the automated preparation and analysis of amino acids, sugars and organic acids in grape juice and wines”. First, liquid samples were prepared and cleaned using ASTED (Gilson, Villiers-le-Bel, France). This consists of a large capacity XYZ autosampler, a Model 402 dilutor (equipped with two 1-ml syringes), a flat-bed dialyser (cellulose acetate membrane with 15 kDa mol. wt. cut-off, 100 μl donor channel, 175 μl recipient channel) and two sixport Model 7010 valves, fitted one with a 20 μl injection loop. The samples and reagent racks were subjected to thermostat at 4 °C using a Model 832 temperature regulator. This was controlled from a keypad using 722 (version 2.0) software. The ASTED was coupled to an HPLC system which consists of two pumps of Model 306 with 5SC pump heads; a manometric module of Model 805, and a mixer module for gradient elution of Model 811C. There are also a Model 831 temperature regulator (Model 831), a fluorometer (Model 122) and, a UniPoint (version 1.4) System Software that functions for control and data handling. Finally, the derivatized amino acid was analyzed on a Hypersil BDS C18 column (3 μm; 150 ´ 4.6 mm) from Hypersil (Runcorn, UK) (Linget, Netter, Heems, & Vérette, 1998).
Cakir, E., Ozcan, O., Yaman, H., Akgul, E.O., Bilgi, C., Erbil, M.K. et al. (2005). Elevated plasma concentration of asymmetric dimethylarginine that is reduced by single dose testosterone administration in idiopathic hypogonadotropic hypogonadism patients [Electronic version] The journal of clinical endocrinology & metabolism 90;3 1651-1654.
Healthnotes. (2004). Arginine. Retrieved May 8, 2007 from the healthnotes web site: www.healthnotes.com.
Linget, C., Netter, C., Heems, D., & Vérette, E. (1998). On-line dialysis with HPLC for the automated preparation and analysis of amino acids, sugars and organic acids in grape juice and wines [Electronic Version]. Analusis, 26, 35-39.
Venta, R. (2001). Year-long validation study and reference values for urinary amino acids using a reversed-phase HPLC method [Electronic Version]. Clinical Chemistry 47:3 575–583.
Wu, G., Knabe, D.A., & Sung, W.K. (2004). Arginine nutrition in neonatal pigs [Electronic Version]. American Society for Nutritional Sciences.