Experiment: Quantitative Analysis of Caffeine and Metabolites of Caffeine in Saliva
Purpose of Experiment
The purpose of the experiment is to perform quantitative analysis of caffeine and metabolites of caffeine in Saliva. The quantitative analysis would aid in determining the clearance rate of caffeine as well as qualify the rate of metabolism of caffeine to its metabolites, mainly paraxanthine. This rate of metabolism can be determined by measuring the amount of metabolites (usually paraxanthine) in the saliva.
Need to Study Caffeine
Variability
Caffeine is metabolized by a specific form of isozyme that belongs to the cytochrome p450 family of enzymes. This specific isozyme is CYP1A2 (Dews, 2012). CYP1A2 is a biological protein which is produced by hepatocytes. This implies that it CYP1A2 is coded by a specific gene. Humans exhibit genetic variability and this means that there exists variability in the CYP1A2 isozyme among people. Thus, the CYP1A2 isozyme of different person metabolizes caffeine at different rates. Moreover, the rate of caffeine metabolism by CYP1A2 differs in the same person depending on time after caffeine consumption, physiological status, health and status of liver functions (Guest et al., 2015). Thus, there is need to study caffeine variability so as to measure the rate of caffeine metabolism as well as qualify the physiological (and health) status of an individual. Also, one would be able to determine, measure, and quantify the amounts of the metabolites of caffeine that are in the body. These metabolites are paraxanthine, theobromine and theophylline.
Additionally, understanding the rate of caffeine metabolism would enable one to predict or determine the onset of the physiological effects caused by the metabolites; paraxanthine, theobromine and theophylline (Dews, 2012). These physiological effects include increased rate of lipolysis, increased volume of urine output, increase in heart rate, relaxation of bronchial smooth muscles, and dilatation of blood vessels (Guest et al., 2015). Thus, the study of variability in caffeine metabolism would enable a physician to predict if a drug containing caffeine can be used to manage asthma depending on whether the patient is a rapid metabolizer or slower metabolizer of caffeine.
Methodology
Saliva is a bio-fluid which contains both organic (including immunoglobulin) and inorganic matter (Dews, 2012). For this experiment, there is need to remove the large proteins (organic matter) from the saliva so as to eliminate organic compound that will interfere with the experiment and thus lead to the production of false results. For this reason, ammonium sulphate is used to precipitate immunoglobulin. This reduces the density of the resulting analyte. The addition of the internal standard serves to calibrate the response of the saliva to ammonium sulphate and thus improve the precision of the quantitative analysis experiment. The internal standard used in this experiment is acetaminophen as it provides the slightly alkaline environment that is optimum for the reaction between the analyte and chloroform (Gilbert, 2013).
The 6th test tube which only contains the internal standard serves as the control in the experiment. The control would provide the standard …
