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Supplementary Methods

Measurement of PON1 protein levels

In brief, 96-well plates (NUNC Immunosorb Maxisorb; Nalge Nunc Int., Rochester, NY) were coated overnight with a serum standard generously provided by Dr. Mackness, or with unknown serum diluted 1:2000 in 50mM sodium carbonate/bicarbonate solution. After washing in PBS with 0.1% BSA and incubating for one hour in PBS with 1% BSA, the plates were incubated for one hour with 100 ml of commercially available rabbit anti-PON1 antibody (1:500, Sigma, St. Louis, MO), washed in PBS 0.1% BSA and incubated for one hour with 100 ml of HRP-conjugated anti-rabbit secondary (1:5000, Jackson ImmunoResearch, West Grove, PA) and developed with 100 ml of 1mM tetramethylbenzidine/0.03% hydrogen peroxide in 0.1M citrate buffer (pH 4.2). The reaction was stopped with 50 ml of 2N sulfuric acid and read at 420 nm (with reference 590 nm). Samples were run in duplicate and averaged across three different experiments.

Paraoxonase, Diazoxonase, and Arylesterase assays

Paraoxon and diazoxon were obtained from Chem Service (West Chester, PA). Phenylacetate was obtained from Sigma Chemical (St. Louis, MO). Rates of paraoxon, diazoxon, and phenylacetate (arylesterase) hydrolysis were determined in a SPECTRAmax® PLUS Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA) using either Ultraviolet transparent 96 well microplates from Costar (Cambridge, MA) for UV readings (270 nm), or standard flat bottom 96 well microplates from Greiner One (Monroe, NC) for visible wavelength (405 nm). All assays were carried out in triplicate using a multichannel pipettor (Matrix, Hudson NH). Outliers were repeated. Rates of hydrolysis were measured for 4 minutes with only initial linear rates used for calculations, and results were normalized using pathlength correction.

Serum was diluted 1:10 in dilution buffer (9 mM Tris-HCl, pH 8.0, 0.9 mM CaCl2). The diluted serum samples (20 ml) were assayed for paraoxonase (POase) by addition of 200 ml 1.2 mM paraoxon substrate in assay buffer (2M NaCl, 0.1M Tris-HCl, pH 8.5, 2.0 mM CaCl2). Rates of serum paraoxon hydrolysis were monitored at 405 nm at 37oC. Measurement of cerebrospinal fluid (CSF) paraoxon hydrolysis used 150 ml of undiluted sample, with 150 ml of 2X concentrated paraoxon added at 37oC. Activities are expressed in Units/liter, based on the molar extinction coefficient of 18 mM-1cm-1 for p-nitrophenol.

Serum diazoxon hydrolysis (DZOase) was measured by adding 200 ml of 1.0 mM diazoxon substrate in assay buffer to 20 ml of 1:10 diluted serum as above. Rates were monitored at 270 nm at 25oC. Activities are expressed in Units/liter, based on the molar extinction coefficient of 3 mM-1cm-1 for the diazoxon hydrolysis product, 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMHP).

Serum samples were assayed for arylesterase by diluting samples 1:80 in dilution buffer. The assay was initiated by the addition of 200 ml of 3.26 mM phenylacetate in dilution buffer to the samples (20μl). Rates of serum phenylacetate hydrolysis were monitored at 270 nm at 25oC. Assays of phenylacetate hydrolysis by cerebrospinal fluid (CSF) used 25 ml of undiluted sample. Arylesterase activities were expressed in Units/ml, based on the molar extinction coefficient of 1.31 mM-1cm-1 for phenol.