Metabolism
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copyright © 2008 - 2015 David A Bender
Experiments with isolated liver cells:
the citric acid cycle and warming
up post-operative patients
Studies with isolated hepatocytes
Isolated
liver parenchymal cells (hepatocytes) provide an extremely convenient system
for metabolic investigations. The isolated cells are prepared by perfusion of
the liver in situ with collagenase; as the collagen in connective tissue
is hydrolysed, so the cells can be separated by gentle pressure, and suspended
in buffer for incubation. A relatively large number of experiments can be performed
using the cells from the liver of each rat or mouse.
For experiments using radioactive substrates, incubations are normally performed in a conical flask with a small centre well that is sealed with a rubber stopper.
At the end of the incubation perchloric acid is injected into the incubation medium, to denature proteins, disrupt cells and drive off carbon dioxide. The carbon dioxide is trapped by an alkali injected into the centre well, and its radioactivity can then be determined. The acidified incubation medium can be used for the determination of both the concentration and radioactivity of metabolites.
(Note that dpm is a measure of radioactivity = radioactive disintegrations per minute).
The key experiments
which led to elucidation of the citric acid cycle (the tricarboxylic acid cycle,
sometimes called the Krebs' cycle) were described by Krebs and Johnson in 1937.
The photograph on the right shows Sir Hans Krebs with the Warburg manometers
that were used to measure oxygen consumption in these experiments.
Krebs and Kohnson measured the consumption of oxygen by a preparation of minced
pigeon breast muscle incubated with and without the addition of 3 mmol citrate.
The results show the volume of oxygen consumed during the incubation by 460
mg wet weight of tissue.
The complete oxidation of 1 mmol of citrate to carbon dioxide and water consumes 100 µL of oxygen).
µL oxygen consumed |
|||
minutes incubated |
no added substrate |
+ 3 mmol citrate |
difference |
30 |
645 |
682 |
+ 37 |
60 |
1055 |
1520 |
+ 465 |
90 |
1132 |
1938 |
+ 806 |
[From data reported by Krebs HA & Johnson WA. The role of citric acid in intermediate metabolism in animal tissues. Enzymologia 4: 148-156 1937]
What substrate is being oxidised when the minced muscle tissue is incubated
with no added substrate?
They were using freshly prepared muscle tissue, which will have contained a considerable amount of glycogen; this is what is providing the substrate for glycolysis and onward oxidation.
What conclusions can you draw from these results?