Hp series 1100

Plasma levels of NO-related metabolites, including l-citrulline (the precursor of l-arginine), l-arginine (substrate for NO synthesis), ADMA, and SDMA were measured using HPLC (HP series 1100; Agilent Technologies Inc., Santa Clara, CA, USA) with the OPA-3MPA derivatization reagent [42 (link)]. Homoarginine (Sigma-Aldrich, St. Louis, MO, USA) was used as the internal standard.

Plasma L-citrulline (a precursor of L-arginine), L-arginine (the substrate for NO synthase), ADMA, and SDMA levels were measured using HPLC (HP series 1100, Agilent Technologies, Inc., Santa Clara, CA, USA) with the O-phthalaldehyde/3-mercaptopropionic acid (OPA/3MPA) derivatization reagent as we described previously [36 (link)]. According to a protocol validated in our lab [21 (link)], plasma creatinine level was also determined by HPLC.

Plasma levels of citrulline, arginine, ADMA, and SDMA were determined using high-performance liquid chromatography (HPLC: HP series 1100, Agilent Technologies, Inc, Santa Clara, CA, USA). O-phthaldialdehyde 3-mercaptopropionic acid (OPA-3MPA) was used for the fluorescent derivatization [44 (link)]. The standards contained arginine and citrulline at 1–100 μmol/L. For ADMA and SDMA, the standards were set at 0.5–5 μmol/L.

The levels of several components of the NO pathway, including l-citrulline, l-arginine, ADMA, and SDMA (an isomer of ADMA), were measured using HPLC (HP series 1100; Agilent Technologies Inc., Santa Clara, CA, USA) with the O-phtalaldehyde-3-mercaptoprionic acid derivatization reagent [7 (link)]. Standards contained concentrations of 1–100 mM l-citrulline, 1–100 mM l-arginine, 0.5–5 mM ADMA, and 0.5–5 mM SDMA.

Plasma and kidney l-arginine, l-citrulline, ADMA, and symmetric dimethylarginine (SDMA, a stereoisomer of ADMA) levels were measured using HPLC (HP series 1100, Agilent Technologies, Inc., Santa Clara, CA, USA) with the OPA-3MPA derivatization reagent as we described previously [10 (link)]. Standards contained l-arginine, l-citrulline, ADMA, and SDMA in the range of 1–100 μM, 1–100 μM, 0.5–5 μM, and 0.5–5 μM, respectively. The recovery rate was between 90 and 105%. The tissue concentration was factored for protein concentration, which was represented as μmol/mg protein. Plasma and urine creatinine (Cr) levels were analyzed by HPLC as described previously [10 (link)]. The creatinine clearance (CCr) was calculated by dividing the total amount of Cr excreted in urine by the Cr concentration in plasma. CCr values were normalized with respect to body weight.

Blood creatinine and uric acid levels were determined by high-performance liquid chromatography (HPLC, HP series 1100; Agilent Technologies Inc., Santa Clara, CA, USA) according to methods described previously [36 (link)]. The levels of several components of the NO pathway, including l-citrulline, l-arginine, ADMA, and SDMA, were measured using HPLC with the O-phtalaldehyde-3-mercaptoprionic acid derivatization reagent [22 (link)]. Standards contained concentrations of 1–100 mM l-citrulline, 1–100 mM l-arginine, 0.5–5 mM ADMA, and 0.5–5 mM SDMA. The recovery rate was around 95%.

Direct HPLC method was used to measure the MccJ25 encapsulated liposomes. The samples were analyzed with a HPLC system (Agilent HP series 1,100) equipped with an analytical C18 reverse phase column (250 × 4.6 mm Phenomenex Luna Å) at 40°C and a diode array detector (DAD) at 214.8 nm wavelength. Solvent A (1L of HPLC water acidified with 416 μL HCl) and Solvent B (HPLC-grade acetonitrile). The samples (100 μL) were injected and eluted at a flow rate of 1.5 mL/min with a linear gradient of solvent B from 0 to 40% in 40 min. The encapsulation efficiency (% EE) was measured by comparing the quantities of MccJ25 before and after encapsulation.