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The addition of an alcohol disrupts the hydrogen bonds that protect the central methylene group, which then reacts with the diazo dye (3,5-dichlorophenyl diazonium), along with conjugated bilirubin, thus measuring total bilirubin in a strongly acidic medium (pH 1-2)

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The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript