Effects of Chemical Structure on Drug Metabolism - Chemistry Assignment Help

Assignment Task:

Introduction

The processes of metabolism and excretion are mainly responsible for terminating the action of drugs in the human body. The metabolic changes to which drugs are subjected in vivo are directed primarily towards the formation of metabolites which have physicochemical properties favourable to their excretion. The majority of drugs are metabolised in the liver and the enzymes responsible for these biotransformations can occur in the soluble, mitochondrial or microsomal fractions of this tissue. Two types or phases of biotransformation can be distinguished: Generally, Phase I reactions (oxidations, reductions and hydrolysis) are concerned with either the unmasking or introduction of small functional groups into the molecule being metabolised. These reactions may be accompanied by fragmentation leading to the formation of small molecules such as carbon dioxide and formaldehyde which may be excreted in exhaled air from the lungs or utilised in other biochemical reactions in the body.

Phase II, or conjugation reactions, can follow phase I reactions by the addition of polar endogenous molecules to the functional groups, leading to the formation of water-soluble conjugates such as glucuronides, sulphates and mercapturates. Many drugs undergo a phase I reaction followed by a phase II reaction, although a phase II reaction can occur if the drug already has a functional group (e.g. the p-hydroxy group of paracetamol forms a glucuronide or sulphate).

Experimental

You will work individually and determine the rate of metabolism of each designated substrate.

1. Label 15 Eppendorf tubes and place them to cool in ice trays. Also label 15 tubes for step 9. Label tubes with your initials and numbered 1-15. Prepare 2 sets of tubes.

2. Add 150μl of 15g% w/v ZnSO4 (a stop solution which will inhibit demethylation) to the first 3 tubes (1-3). The reaction mixtures in these tubes will serve as blanks.

3. Add 300μl of the liver homogenate to each tube (1-15).

4. Add 300μl of the NADPH regenerating solution / semicarbazide / substrate cocktail to each tube. For the blanks use the cocktail containing m-hydroxyanisole (1-3); for tubes 4-5 use the cocktail containing your first test substrate; for tubes 6-7 use the cocktail containing your second test substrate etc. so that you have a blank in triplicate and duplicates of each of your 6 tests.

5. Cap and vortex mix your tubes (1-15) and incubate them for 30 min in a water bath at 37°C (Do NOT put them in the 60°C water bath by accident).

6. Add 150μl 15g% w/v ZnSO4 to each tube (4-15) except the blanks to stop the reaction.

7. Add 150μl of saturated BaOH to each tube (1-15) to precipitate proteins and sulphates and vortex mix.

8. Stand the tubes on ice for 20 min during which time a white precipitate should form and then centrifuge the tubes at 13,000 rpm for 2 min.

9. Transfer 240μl of clear supernatant from each tube into the clean labelled tubes, add 60μl of NASH reagent to each, cap the tubes and incubate them carefully in a water bath at 60°C for 30 min to develop the coloured reaction product (yellowish colour).

10. Cool the solutions to room temperature on ice and then repeat the centrifugation at 13,000 rpm for 2 min. If there is a precipitate in the tubes at this point, remove the supernatant into a clean tube and respin as before. Transfer 250μl of supernatant from each tube into individual wells of a microtitre plate. 

11. Measure absorbance of the wells at 405 nm to determine reaction product and 620nm to correct for inter-well differences.

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