UA (purity >99%) was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A). [1,3-15N2]-UA (purity >98%) was used as the internal standard (ISTD) and it was purchased from Cambridge Isotope Laboratories (Andover, MA, U.S.A). Ammonium acetate, acetic acid, potassium hydroxide (KOH), and methanol were obtained from Fisher Scientific (Pittsburgh, PA, U.S.A). Trichloroacetic acid (TCA) was purchased from LabChem Inc (Pittsburgh, PA, U.S.A). HEPES buffer was obtained from Mediatech (Hernond, VA). The sample was filtered through 0.22 μm Nylon centrifuge tube filter (COSTAR Corning Inc, NY, U.S.A). Fresh frozen human plasma was purchased from Civitan Regional Blood Center (Gainesville, FL, USA).
2.2. Preparation of standard solutions
Stock solution of UA was prepared at a concentration of 10 mM in 0.3 M KOH. The working solutions of various concentrations (0.1 and 1.0 mM) were prepared with 0.3 M KOH. All stock and working solutions were kept below 4°C. The ISTD was prepared by diluting a [1,3-15N2] UA stock solution at 1.0 mM with 0.3 M KOH. 10 % TCA was kept in the refrigerator before use.
2.3. Urine sample collection and preparation for UA analysis
Urine samples were stored at −80°C before analysis. For the analyses of urinary UA, urine samples were diluted 8-fold by volume with distilled water. The ISTD 1,3-15N2-UA was added at a final concentration of 0.24 mM. 4 μL of internal standard and 200 μL 10 % TCA (w/v) were added to 1 mL diluted urine sample. This solution was vortexed for 30s and then filtered through a 0.22 μm Nylon filter and the filtrate was loaded into an autosampler vial and analyzed by LC-MS/MS.
2.4. Plasma sample collection and preparation for UA analysis
Plasma samples were stored at −80°C until analysis. In the case of UA analyses, each plasma sample (500 μL), which included 0.24 mM of 1,3-15N2-UA as the ISTD, was treated with 100 μL 10 % TCA (w/v). The sample was vortexed and filtered through a 0.22 μm filter by centrifugation at 14,000 rpm. The filtrate was loaded into an autosampler vial and analyzed by LC-MS/MS.
2.5. Cell culture and cell lysate preparation
Human umbilical vein endothelial cells (HUVECs, Clonetics) were plated on 60-mm culture plates and maintained at 37°C and 95% O2/5% CO2 in EBM BulletKit® Media (Clonetics). After cells grew at 90 % confluence, cells were then washed with PBS and trypsinized. The cell number was calculated by hemocytometer. Cells were lysed by with 0.3 M KOH, and then the lysate was sonicated. After the total volume, including total cell volume and volume of KOH solution, was calculated, the internal standard, 1,3-15N2-UA, was added to final concentration of 0.17 mg/dl. The samples were centrifuged at 136,000 rpm for 30 min and the supernatant was filtered through a 0.22 μm Nylon filter at 3,000 rpm for 10 minutes. The filtrates were stored at −80°C for the measurement of UA by LC-MS/MS.
2.6. Instrumentation and LC-MS/MS conditions
The LC-MS analyses were carried out with a ThermoFinnigan Surveyor liquid chromatography system (ThermoFinnigan, San Jose, CA, USA) and a TSQ Quantum Discovery triple quadrupole mass spectrometer (ThermoFinnigan, San Jose, CA, USA) equipped with an ESI interface operated in negative-ion mode detection. In the TSQ Quantum instrument, nitrogen was used for both the sheath and auxiliary gases at 60 and 20 arbitrary units, respectively. The heated capillary temperature was maintained at 300°C. The collision pressure was 1.5 mTorr and the collision energy was 25V. The operation of the LC-MS and data analysis was performed using the ThermoFinnigan Xcalibur 1.4 software.
Liquid chromatography analyses were performed in a gradient elution mode using Phenomenex Luna 5μ C18(2) 100Å (150 mm × 4.6 mm) column (Phenomenex, Torrance, CA, USA) coupled with a Phenomenex Luna C18 (2), 5 μm particle size guard column. The mobile phase used included 5 mM ammonium acetate/0.1 % acetic acid (A) and methanol (B). The mobile phase flow was 0.6 mL min−1 and the flow was split (1:3) prior to the MS. The injection volume was 20 μL. The gradient began at 95% A. The composition was linearly ramped to 25% B over the next 4.5 min, remained constant for 1.5 min, then reversed to the original composition of 95% A over 0.5 min, after which it was kept constant for 0.5 min to re-equilibrate the column. UA was analyzed in the negative ESI mode and the parent ion of UA was m/z 167.0 and monitored SRM ions were m/z 124.0 and 96.0. In case of ISTD the parent ion of 1,3-15N2-UA was m/z 169.0 and monitored SRM ions were m/z 125.0 and 97.0.
The quantification of UA in vivo and in vitro was carried out against a calibration curve prepared in each of the matrices. The UA standard concentrations were 1.0, 4.0, and 16.0 mg/dl for urine and plasma calibration curves. In the case of cell lysates, calibrators were applied at the concentrations of 0.0084, 0.017, 0.084, 1.000, and 4.000 mg/dl of UA. The ISTD 1,3-15N2 UA was added at a final concentration of 4.0 mg/dl in urine and plasma and 0.17 mg/dl in cell lysate. The calibration curves were plotted as concentration vs. peak area ratio of the analytes and the ISTD.
2.8. Validation of the analytical method
Precision of the method was determined by calculating the intra-day and inter-day coefficients of variation (C.V. %) for UA. To measure the intra-day variance, five sets of prepared samples at each concentration level plus fixed concentration of internal standard were generated by using the urine and plasma or water stock solutions of the analytes. The intra-day variance was calculated based on five trial measurements accomplished in the first day, and the inter-day variance was calculated based on the results of five analyses carried out on five consecutive days. Accuracy of the method was determined by comparing measured concentrations with those added concentrations, and was expressed as RE (relative error, %). The calibration range for validation were as follows: 1) the range of 1.0 –16.0 mg/dl including 4.0 mg/dl of 1,3-15N2-UA as ISTD in urine and plasma or water stock solutions, and 2) the range of 8.40 μg/dl - 4.00 mg/dl including 0.17 mg/dl of ISTD in cell lysate and water stock solutions. The recovery test for the protein precipitation was conducted using corresponding concentrations in water compared with samples spiked with UA in plasma, urine or cell lysates treated with 10 % TCA. For absolute values in each matrix, we first measured matrix samples of each matrix. The plasma and urine pools contained 4.46 mg/dl and 5.20 mg/dl of UA, respectively and these UA values were subtracted from the values of the matrix samples that had been spiked with isotope-labeled UA. This subtracted value was compared with the corresponding levels of UA in water samples.
2.9. Sample collection for normal plasma and urine
Fresh frozen human plasma (n=10) was purchased from Civitan Regional Blood Center (Gainesville, FL, USA). Subjects who provided urine samples included n=26 normal subjects from Baylor College of Medicine. Sample collection was approved by the local Institutional Review Boards for the Baylor College of Medicine and the University of Florida, respectively.
2.10. Measurement of intracellular UA levels in kidney proximal tubular cells treated with fructose
Human kidney proximal tubular cells (HK-2), an immortalized cell line from normal adult human kidney [32
], were obtained from American Type Culture Collection (ATCC, Rockville, MD). Cells were grown to confluence in Dulbecco’s Modified Eagle Medium:Nutrient Mix (50/50) F-12 (DMEM/F-12) with L-glutamine and HEPES buffer (Mediatech, Hernond, VA). Cells were cultured at 37°C in 95% air and 5% carbon dioxide (CO2
) to 70–75% of confluence. Cells were harvested on ice in a buffer containing 25 mM HEPES (pH 7.1), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA and homogenized by 100 strokes of the pestle. 5 mM D-fructose was added to the cells to induce UA production and time intervals were 7 points (1, 5, 30, 120, 180, 1440, and 4320 min) after treatment of the cells with 5 mM D-fructose. The cells were rinsed with ice-cold PBS and harvested on ice in the buffer containing 25 mM HEPES (pH 7.1), 100 mM KCl, 1 mM DTT and 0.1 mM EDTA. The cell extracts were homogenized by 100 strokes of the pestle and frozen/thawed 4 times in liquid nitrogen. All samples were stored at −80°C prior to analysis. Before the analysis, they were thawed in a cold water bath and filtrated through a 0.2 μm Micro Centrifuge Nylon filter by centrifugation for 10min at 14,000 rpm. The filtrates were sealed in amber glass vials using Teflon-lined caps for LC-MS/MS analyses. The calibrators were 0.0084, 0.017, 0.084, 1.000, and 4.000 mg/dl UA.