metabolic pathway

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EXPERIMENT

Metabolic pathway analysis using radioactive isotopic tracers intake
Aishwarya Mishra

Introduction:
Physiological, pathological and pharmacological alterations in energetic substrate metabolism have been known to contribute to effective functioning of an organ. Hence, pharmacological interventions have been shown to improve clinical symptoms of certain pathologies, such as ischemic heart disease. In order to manipulate metabolism, however, one has to be able to correctly measure and know the contribution of a particular pathway to energy production. Many methods have been developed and adapted to a wide range of techniques. Radioactive substrates have been utilized for many laboratories with success. We adapted, from well-established radioactive techniques, measurements for glycolysis and glucose oxidation in C2C12 myocytes.

Recently, increased production of stable
isotopically labelled
compounds and dramatic advances in instrumental systems, for their measurement have led to a resurgence of interest in application of nonradioactive isotopic tracer techniques to biomedical problems in vivo. Concomitantly, an increasing concern over the administration of radioisotopes to human subjects has further spurred rapid advancement of the stable isotopic field.

Aim:
To study the rate and mechanism of metabolism pathways using radioactive isotopic tracers
Theory:
Manipulation of energetic substrate metabolism as a potential therapeutic intervention is a new and attractive approach to treat heart disease. Precise measurement of metabolic parameters is crucial when evaluating the rate of the pathway. This is complicated due to limitations in creating the physiological similarities with all hormones and substrates for a specific tissue. Metabolic measurements are possible thanks to the use of radioactively-labelled substrates [1, 2]. Muscle cells normally drive most of their energy from the oxidation of fatty acids and carbohydrates. Glycolysis also provides a smaller amount of energy. Two by-products of these pathways are H2O and CO2. To quantify the amount of 3H2O or as a measure of a pathway is possible if only the experimenter knows at which step of the pathway these molecules are produced. To measure glycolysis, for example, the experimenter uses

[5-3H]-glucose. The radioactive label on glucose molecule is released as 3
H2O at the enolase step of glycolysis. As shown in the figure below.

When measuring glucose oxidation, the experimenter should use [U14C-] glucose and collect 14CO2 that is either released into the air or trapped in the medium as carbonate. 14CO2 is produced at the step where pyruvate is metabolized to acetyl CoA and by citric acid cycle. As shown in the diagram below:

Materials:
 C2C12 mouse myoblasts
 Dulbecco's growth medium,
 bovine serum
 Penicillin G
 Streptomycin
 Glasswares














Reaction vessel for incubation
Sonicator
Haemocytometer
[5-3H]-glucose
[U-14C-]-glucose
KOH or benzothonium hydroxide
Scintillation counter
Incubation plates
Filter paper
KOH
HClO4
Microscope

Procedure:
The lab procedure is planned over a span of 10-11 days. The initial groundwork requires the transformation of myoblasts in the growth medium. The initial day lab requires growth of the myoblasts and initiation of their differentiation. The following protocol is followed for transformation:

 Take Dulbecco's modified medium in a conical vessel of 25 ml according to the mouse myoblasts quantity.
 To the medium, add 10% foetal bovine serum(FBS)
 Add 100 U/ml of Penicillin G
 Add 100 g/ml of Streptomycin(Growth medium)
 C2C12 Mouse myoblasts is added to the medium for differentiation.  The medium is allowed to reach 80% confluency.
 The cells are then differentiated in the same medium that contained 1% FBS (differentiation medium).
 Visual confirmation of myotube formation of all myoblasts in the differentiation medium is...
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