Fig 1.
Schematic outlining L-arginine metabolism via nitric oxide synthase and arginase pathways.
L-arginine is metabolized via two major enzymatic pathways. In the nitric oxide pathway, L-arginine is catalyzed by nitric oxide synthase (NOS), in the presence of essential cofactors, to produce nitric oxide (NO) and L-citrulline. Alternatively, L-arginine is hydrolyzed by arginase to yield L-ornithine and urea.
Fig 2.
Schematic diagram outlining L-arginine supplementation of medium for BNL CL2 or 3T3 L1 cell growth.
Cells were maintained in SILAC L-Arg deficient media supplemented with 0, 400 or 800 µM L-arginine or in control complete DMEM containing 250 µM L-arginine (2 mL/ well). Samples were collected at T = 0 (untreated, the cells were maintained in complete DMEM media for 24 h prior to the L-Arg addition experiment) and 24 and 72 h post addition of L-arginine for analysis.
Fig 3.
Chemical reaction between OPA and primary amino acid to form an adduct (OPA-ME-AA).
Primary amino acid (AA) forms a derivative (OPA-ME-AA) with o-phthalaldehyde (OPA) in the presence of a thiol, 2-mercaptoethanol (ME). The adduct is well-resolved by gradient elution HPLC. The reaction takes place rapidly at room temperature at alkaline pH.
Table 1.
HPLC gradient program for separation of L-arginine, L-citrulline and L-ornithine at flow rate 1.1 mL/min.
Table 2.
LC-MS gradients used to identify the derivatisation adducts of amino acids (OPA-ME-AA) at a flow rate 1.5 mL/min.
Table 3.
Comparison of expected mass of the adducts with the experimental mass.
Fig 4.
LC chromatogram with the signals observed for L-arginine with OPA and mass spectrum for OPA in water.
Three signals appeared for L-arginine and OPA reagent in the LC trace (A, top panel). The two signals on the left are combination products of OPA and a signal on the right is for the OPA-ME-L-arginine adduct. Signals for OPA in water alone on the LC appeared as two peaks (A, bottom panel). Mass spectra data for OPA in water (B: 1, 2 and 3) shows multiple combination products are generated from OPA.
Fig 5.
Schematic reaction mechanism and mass spectrum for formation of L-arginine adduct in positive polarity.
(A) Reaction schematic for formation of L-arginine adduct (isoindole derivative) and expected mass with positive polarity [MH]+ by reacting with the derivatizing agent OPA containing ME. (B) LC chromatogram with the signals observed for L-arginine with OPA absorbance at 220 nm. The two peaks on the left are derived from OPA or other products of OPA and the single peak on the right is for the expected OPA-ME-L-arginine adduct. (C) Mass spectra with a protonated molecule detected at a base peak at m/z 351.1 for the diagnostic fragment ion for the OPA-ME-L-arginine adduct.
Fig 6.
Schematic reaction mechanism and mass spectrum for formation of L-arginine adduct in negative polarity.
(A) Reaction schematic for formation of L-arginine adduct (isoindole derivative) and expected mass with negative polarity [M-H]- by reacting with the derivatizing agent OPA containing ME. (B) LC chromatogram with the signals observed for L-arginine with OPA absorbance at 220 nm. The two peaks on the left are derived from OPA or other products of OPA and the single peak on the right is for the expected OPA-ME-L-arginine adduct. (C) Mass spectra with an ionized molecule detected at a base peak at m/z 349.0 for the diagnostic fragment ion for the OPA-ME-L-arginine adduct.
Fig 7.
Schematic reaction mechanism, HPLC chromatogram and mass spectrum for formation of L-citrulline adduct in positive polarity.
(A) Reaction schematic for formation of L-citrulline adduct (isoindole derivative) and expected mass with positive polarity [MH]+ by reacting with the derivatizing agent OPA containing ME. (B) Mass spectra showing a protonated molecule detected at a base peak at m/z 351.0 for the diagnostic fragment ion for the OPA-ME-L-citrulline adduct.
Fig 8.
Schematic reaction mechanism, HPLC chromatogram and mass spectrum for formation of L-ornithine adduct in positive polarity.
(A) Reaction schematic for formation of L-ornithine adduct (isoindole derivative) and expected mass with positive polarity [MH]+ by reacting with the derivatizing agent OPA containing ME. (B) Mass spectra showing a protonated molecule detected at a base peak at m/z 309.1 for the diagnostic fragment ion for the OPA-ME-L-ornithine adduct.
Fig 9.
Relative and absolute quantification of residual serum L-Arg, L-Cit and L-Orn in BNL CL2 cells cultured in different L-Arg concentrations and the control for 24 and 72 h.
Relative (A), (C) and (E), respectively) and absolute (B), (D) and (F), respectively) residue amount of L-Arg, L-Cit and L-Orn analysed in culture supernatant of BNL CL2 cells cultured in different amount of L-Arg (0, 400 and 800 µM) in L-Arg deficient media and the control complete DMEM media (containing 250 µM L-Arg) for 24 and 72 h. Untreated samples were maintained in complete DMEM media (containing 250 µM L-Arg) and collected 24 h after the incubation prior to L-Arg addition experiment (T = 0). Data points represent the mean ± SD of each sample. Error bars represent the standard deviation from the mean (n = 3). Tables summarise two-way ANOVA followed by a Tukey multiple comparison test using GraphPad Prism 9.4.1. The stars indicate the levels of significance; ns = P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001 and **** = P ≤ 0.0001.
Fig 10.
Relative and absolute quantification of residual L-Arg, L-Cit and L-Orn in culture medium of 3T3 L1 cells cultured in different L-Arg concentrations and the control for 24 and 72 h.
Relative (A), (C) and (E), respectively) and absolute (B), (D) and (F), respectively) residue amount of L-Arg, L-Cit and L-Orn analysed in culture supernatant of 3T3 L1 cells cultured in different amount of L-Arg (0, 400 and 800 µM) in L-Arg deficient media and the control complete DMEM media (containing 250 µM L-Arg) for 24 and 72 h. Untreated samples were maintained in complete DMEM media and collected 24 h after the incubation prior to L-Arg addition experiment (T = 0). Data points represent the mean ± SD of each sample. Error bars represent the standard deviation from the mean (n = 3). Tables summarise two-way ANOVA followed by a Tukey multiple comparison test using GraphPad Prism 9.4.1. The stars indicate the levels of significance; ns = P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001 and **** = P ≤ 0.0001.