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5 protocols using barium hydroxide

1

Analytical Quantification of Adducts

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All chemicals were ACS grade or higher, purchased from Sigma Aldrich (St Louis, MO): styrene (substrate), styrene oxide (product), styrene glycol (product), benzyl alcohol (internal standard), acetaminophen (substrate), and 2-acetoamidophenol (internal standard). Barium hydroxide, zinc sulfate, 1,4-dioxane, HPLC grade methanol and HPLC grade acetonitrile were purchased from Fisher Scientific, Wilmington, MA. The acetaminophen-glutathione (APAP-GSH) and acetaminophen-cysteine (APAP-cys) adducts were synthesized in-house. For purification of adducts, solid-phase extraction Oasis MAX columns were obtained from Waters Corp. (Milford, MA). NAPQI solid was provided as a generous gift from Drs. Dean Roberts and Laura James at Arkansas Children’s Hospital (Little Rock, AR).
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2

Isotopic Labeling Protocol for Organic Synthesis

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Millipore water was obtained from Milli-Q
water system. Acetonitrile was purchased from PHARMCO-AAPER (Brookfield,
CT). Formic acid (88%), barium hydroxide, and calcium oxide were obtained
from Fisher Scientific (Fair Lawn, NJ). Metal magnesium turnings,
lithium hydroxide, potassium hydroxide, sodium formate, rubidium chloride,
cesium carbonate, and Formic acid-d2 (95
wt % in D2O, 98 atom % D) were obtained from Sigma-Aldrich
(Saint Louis, MO). Strontium carbonate was obtained from J. T. Baker
(Avantor, Center Valley, PA). H218O was purchased
from Cambridge Isotope Laboratories (Cambridge, MA). All chemicals
were used without further purification.
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Synthesis of Metal Complexes

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Acetonitrile
was purchased from PHARMCO-AAPER
(Brookfield, CT, USA). Formic acid (88%), barium hydroxide, and calcium
oxide were purchased from Fisher Scientific (Fair Lawn, NJ, USA).
Metal magnesium turnings, lithium hydroxide, potassium hydroxide,
sodium formate, rubidium chloride, cesium carbonate, gallium metal,
indium metal, thallium(III) acetate, lead(II) carbonate, antimony(III)
oxide, and Formic acid-d2 (95 wt % in
D2O, 98 atom % D) were purchased from Sigma-Aldrich (Saint
Louis, MO, USA). Strontium carbonate was obtained from J.T. Baker
(Avantor, Center Valley, PA). H218O was purchased
from Cambridge Isotope Laboratories (Cambridge, MA). All chemicals
were used without further purification.
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4

Measuring Intestinal Lactase and Sucrase

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Intestinal lactase and sucrase activities were measured as previously described (33 (link), 34 (link)). Briefly, mucosal homogenates were prepared and incubated with 0.6 M lactose (Thermo Fisher Scientific) or 0.3 M sucrose (Thermo Fisher Scientific) buffer (dissolved in 0.0625 M maleate buffer) for 60 min at 37°C. The reaction was stopped by the addition of 2.0% zinc sulfate (Thermo Fisher Scientific) and 1.8% barium hydroxide (Thermo Fisher Scientific), and the amount of glucose released was detected via a glucose oxidase reagent (Thermo Scientific). Enzyme activity was in μmoles glucose per minute per gram of protein. Protein was measured in intestinal homogenates via the Bradford method (Bio-Rad).
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5

Intestinal Lactase and Sucrase Activities

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Intestinal lactase and sucrase activities were measured as previously described [28 (link),29 (link)]. Mucosal homogenates were prepared and incubated with 0.6 M lactose (Thermo Fischer Scientific, Hanover Park, IL, USA) or 0.3 M sucrose (Thermo Fisher Scientific, Hanover Park, IL, USA) buffer (dissolved in 0.0625 maleate buffer) for 60 min at 37 °C. The reactions were stopped by adding 2.0% zinc sulfate (Thermo Fisher Scientific, Hanover Park, IL, USA) and 1.8% barium hydroxide (Thermo Fisher Scientific, Hanover Park, IL, USA), and the amount of glucose release was detected by using a glucose oxidase reagent (Thermo Fisher Scientific, Hanover Park, IL, USA). Enzyme activity is expressed as µmoles glucose/minute/g protein. Protein was measured in intestinal homogenates using the Bradford method (Bio-Rad, Hercules, CA, USA).
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