Supplemental Data Ms ID# CLINCHEM/2006/07489
Legends for the Scheme and Figures in Supplemental Data
Scheme 1.
Final procedure followed for the analysis of total isoprostanes in plasma samples. In the case of urine samples, only free isoprostanes were measured, by eliminating the initial step (alkaline hydrolysis).
Supplemental Figure 1.
Linearity of the assay with increasing volumes of plasma (A) or urine (B).
Increasing amounts of plasma or urine were analyzed for isoprostane levels as described in the text, after spiking with 200 pg of deuterated iPF2α-III.
Supplemental Data Figure 2.
Linear dynamic range of the assay.
Increasing amounts of unlabeled iPF2α-III were mixed with a fixed amount of deuterated standard (200 pg), and analyzed by LC/MS as described in the text.
Supplemental Data Figure 3.
Correlation of LC/MS values with LC/MS/MS values.
The isoprostane levelsin 5 urine samples were determined after immunoaffinity column chromatography by either the present method (LC/MS) or by LC/MS/MS. The LC/MS/MS was performed with an API 4000 Qtrap machine connected to an Agilent HPLC system. The column used was Zorbax Eclipse XDB C18 column (4.6 X 150 mm). A linear gradient of 75% methanol in water to 100% methanol was employed with both reservoirs containing 5 mg/mL formic acid and 5 mM ammonium formate. The eluting compounds were ionized by ESI, and analyzed by multiple reaction monitoring (m/z 353/193 for iPF2α-III, and m/z 357/197 for the dueterated standard).
Supplemental Data, Figure 4.
Ex vivo oxidation of plasma.
Aliquots (0.3 mL) of plasma were diluted 3-fold with phosphate buffer and oxidized with 50 µM CuSO4at 37 0C for varying periods of time. The oxidation was stopped by the addition of EDTA (0.6 mmol/L), and the isoprostane levels were determined as described in the text. Parallel samples of oxidized plasma were extracted for total lipids, and the conjugated diene concentration measured by absorbance at 234 nm in a spectrophotometer.