ONLINE SUPPLEMENT A PHASE II RANDOMIZED PLACEBO …

ONLINE SUPPLEMENT

A PHASE II RANDOMIZED PLACEBO-CONTROLLED TRIAL OF ORAL N-ACETYLCYSTEINE IN CYSTIC FIBROSIS.

Supplementary Methods. Measurement of GSH/GSSG in whole blood GSH and GSSG in whole blood were determined by high performance liquid chromatography (HPLC) using a boron-doped diamond cell for electrochemical detection (EC) as previously described (Park et al, 2010). Whole blood was deproteinized with 9 volumes of 10% Trichloroacetic acid and supernatant and stored at -80?C until analysis. An aliquot of 10 ?L was injected directly into the HPLC-EC system.

Park HJ, Mah E, Bruno RS. Validation of high-performance liquid chromatography-boron-doped diamond detection for assessing hepatic glutathione redox status. Anal Biochem. 2010; 407(2):151-9.

Measurement of N-acetylcysteine in whole blood. NAC in whole blood was determined by stable-isotope dilution liquid chromatographyelectrospray injection tandem mass spectrometry (LC-ESI-MS/MS). Whole blood was deproteinized with 9 volumes of 10% Trichloroacetic acid and supernatant was stored at -80?C until analysis. The supernatant was further diluted with 2 volumes of mobile phase containing

2?M 2H3-NAC. The sample (5 ?L volume) was was injected into a Shimadzu Prominence LC System interfaced with a 4000 QTRAP? LC-MS/MS (Applied Biosystems). Solvents for HPLC were: A, 4mM ammonium acetate, 0.1% formic acid, 0.1% heptafluorobutyric acid (pH=2.5); B 100% methanol and 0.1% formic acid. Chromatographic separation was achieved on a 2.0mm?250mm EZ-faast column from Phenomenex. Samples were eluted isocratically at a flow rate of 250L/min (A = 25% and B = 75%) with a total run time of 5 minutes. The source operated in positive ion mode at an ion spray voltage of +5000 V. The observed m/z values of the fragment ions were NAC 1 (m/z 164122), NAC 2 (m/z 16476) and 2H3-NAC (m/z 167123). Calibrator (NAC) and internal standard (2H3-NAC) were included in each analytical run for calibration. 1mM stock solution of standard was diluted in distilled water to concentrations of 2000, 1000, 500, 250 nmol/L. All data were collected using Analyst software version 1.4.2.

Measurement of S-nitroso-acetylcysteine (SNOAC) in plasma. For SNOAC measurement, we adapted a previous protocol (Gow et al, 2007). Venous blood was drawn in a 4 ml heparinized (green top) vacutainer tube, which was placed immediately on ice. The vacutainer tube was spun at 400G for 10' in a refrigerated centrifuge and placed in a hypoxic glove box (Coy Laboratory Products, Inc.) at 7% O2 (venous blood oxygen level) for further manipulation. The vacutainer tube was opened and 1 ml of plasma was transferred into a sterile eppendorf. The remainder of plasma and blood cells was reconstituted with 1 ml of sterile ice-cold PBS and set aside for measurement of HIF-1 nuclear translocation (see below). Plasma was spun 3000G at 4?C for 5 minutes in a microcentrifuge and 500 ?l of clear

supernatant was transferred into a Vivaspin 10kDa filter (Sartorius), which was spun at 13000G for 5 minutes. Two aliquots of 100 ?l of the low mass fraction of plasma (eluate having gone through filter) were collected in cryovials and spiked with with 10 ?l of 13C/15N-labeled SNOAC standard. Cryovials were removed from the hypoxic glove box and immediately frozen in liquid nitrogen, and stored at -80?C until LC-MS analysis (storage time < 5 days). For LC-MS analysis, plasma samples were thawed at room temperature, during which a StrataX solide phase extration (SPE) column (60 mg, 3 cc, Phenomenex) was equilibrated with 3 ml methanol and 3 mL 0.1 % formic acid in deionized water by gravity. Valves on the SPE manifold were closed to allow a meniscus of aqueous phase to remain on the SPE sorbent surface. For extraction, 100?l of the sample was applied to the SPE cartridge, valves were open and 2 ml 0.1 % formic acid in deionized water were applied when the sample permeated into the SPE bed. Samples were washed by gravity and vacuum was applied for 15 s to remove the remainder of the solvent. For elution, 0.5 ml of methanol were applied to the cartridge and sample was eluted into a 1.5 ml amber glass HPLC vial using positive displacement pressure from a nitrogen gas line. Eluted sample was diluted by adding 0.5 ml 0.1 % formic acid in deionized water to the vial and immediately loaded into the autosampler for analysis. As a control, 100 ?l of 13C/15N-labeled SNOAC standard were transferred into an amber glass HPLC vial and diluted with 900 ?l of a 50 % methanol 0.1 % formic acid solution. HPLC separation was performed on HP1100 system (Agilent) using isocratic elution conditions (20 % acetonitrile, 0.1 % formic acid, 0.2 ml/min flow rate, Atlantis C18 2.1 x 150 mm 3 ?m column, retention time ~ 3.1 min). MS analysis of SNOAC was performed with a triple quadrupole mass spectrometer (Waters). We used 160 > 60 transition for native SNOAC and 163 > 63 for 13C/15N-labeled SNOAC standard. The 13C/15N-labeled SNOAC standard response was used to determine SNOAC recovery comparing

the ratio of 13C/15N-labeled SNOAC standard in spiked plasma samples and control samples. The concentration of native SNOAC was calculated using a one point 13C/15N-labeled SNOAC standard calibration (assuming that signal response for native SNOAC and 13C/15N-labeled SNOAC standard were the same).

Gow A, Doctor A, Mannick J, Gaston B. S-nitrosothiol measurements in biological systems. J Chromatogr B. 2007; 851:140-51.

Measurement of hypoxia-inducible factor-1 (HIF-1) nuclear translocation in peripheral blood neutrophils. For HIF-1 nuclear translocation measurement, we adapted a protocol detailed elsewhere (Tirouvanziam et al, 2011). In brief, 1 ml of reconstituted whole blood (see beginning of protocol for SNOAC measurement) was mixed with 20 ml of ice-cold 1X Lyze/Fix PhosFlow buffer (BD Biosciences) and incubated overnight at 4?C. The sample was spun at 800G for 10' at 4?C and the leukocyte pellet was resuspended in 1 ml of ice-cold 1X Lyze/Fix PhosFlow buffer and stored at -80?C until batch analysis by image cytometry. For analysis, samples were thawed, washed with PBS-EDTA, permeabilized with Perm I buffer (BD Biosciences), and stained with DRAQ5, cholera toxin B-Pacific Orange (custom conjugate) and a fluoresceinconjugated monoclonal antibody against human HIF-1 (IC1935F, R&D Systems). The combination of cholera toxin B (lipid rafts, enriched in neutrophils) and Draq5 (nuclear staining, enabling gating of live 2N cells) enables also a robust gating of live neutrophils as detailed in Tirouvanziam et al, 2011. After a final wash in Perm I buffer, samples were analyzed on a

ImageStream system (Amnis, Inc.) and the % of nuclear HIF-1 was determined after fluorescence compensation by colocalization with Draq5 staining using the IDEAS software, as previously described (George et al, 2006).

Tirouvanziam R, Diaz D, Gernez Y, Laval J, Crubezy M, Makam M. An integrative approach for immune monitoring of human health and disease by advanced flow cytometry methods, In: Advanced Optical Cytometry: Methods and Disease Diagnoses, Wiley VCH, ISBN 978-3-52740934-1, p. 333- 362 (2011).

George TC, Fanning SL, Fitzgerald-Bocarsly P, et al. Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow. J Immunol Methods. 2006; 311:117-129.

Measurement of vascular-endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) levels in plasma and urine. VEGF and bFGF levels were measured in plasma and urine using ELISA kits (R&D Systems), according to the manufacturer's guidelines.

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