What percentage of the original dose was recovered

Reference no: EM133400526

In addition to glucuronidation, acetaminophen (APAP) can also undergo sulfation to yield the APAP-sulfate conjugate. You need to set up experiments to monitor this reaction in vitro using rat liver cytosol as the sulfotransferase enzyme source. The incubations should contain final concentrations of 0.2 mM APAP, 1 mg/mL cytosol and 1 mM 3'-phosphoadenosine-5'-phosphosulfate (PAPS, the cofactor). Cytosol (20 mg/mL) is commercially available, but the APAP and PAPS stock solutions must be prepared before use. (6 points)

(a) Describe how you would make 10 mL of a 5 mM APAP stock solution (MW = 151 g/mol) in methanol.

(b) PAPS (MW = 507.3 mg/mmol) is expensive (> $100 per milligram!) and you can only afford to buy 2 mg for these experiments. In what volume of buffer should you dissolve this amount to make a 10 mM stock solution?

(c) When everything is ready, you can set up the incubations. If the incubation volume is 1 mL, what volumes of cytosol, APAP and PAPS should you use to get the desired concentrations of each reagent in the incubations? What would be a good control incubation for this experiment?

2.) You are studying the metabolism of a new drug candidate codenamed "K-456". This drug is converted into two metabolites, known as M1 and M2. As part of your investigations, you injected the drug (250 mg/kg) into a rat (200 g) and collected urine (total 10 mL) for 24 hours. Ice cold methanol (10 mL) was added to the urine sample to precipitate the proteins. After centrifugation an aliquot of the supernatant was directly analyzed by HPLC on a C18 column. A standard curve (peak area vs. concentration in mg/mL) for all three compounds was fit to a regression line of y = 1,000x + 17,500.

(a) Using the information in the following table, calculate the amounts (in milligrams) of K-456 and its two metabolites that were present in the original rat urine sample.

Compound Peak Area Retention Time
K-456 450,000 10.3 min
M1 1,200,000 7.1 min
M2 750,000 14.2 min

(b) What percentage of the original dose was recovered in the urine sample? HINT: start by calculating the amount of K-456 (in milligrams) that was administered to the rat.

Reference no: EM133400526

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