Metabolism
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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?
These experiments show a considerably greater increase in oxygen consumption than can be accounted for by the total oxidation of the citrate that was added to the incubation. Complete oxidation of 3 mmol of citrate would consume only 300 µL of oxygen, while over 90 minutes there was additional consumption of 806 µL of oxygen, more than two and a half times more than would be accounted for by the citrate.
Citrate obviously has a catalytic effect, increasing the rate at which other substrates can be oxidised. It was this observation, together with previous studies showing a similar catalytic effect of fumarate, oxaloacetate or succinate, which led Krebs and Johnson to propose a cyclic pathway in which a two carbon compound was added to oxaloacetate to form citrate, then oxidised to yield oxaloacetate again.
At this time the identity of the two carbon compound was unknown, but was assumed to be an acetate derivative. We now know that it is acetyl CoA, which is the end product of both aerobic glycolysis (in cells that have mitochondria) and also beta-oxidation of fatty acids.
Click here for a printable version of the full citric acid cycle
Experiment 1
Isolated hepatocytes were incubated for 40 min in a phosphate / bicarbonate / carbon dioxide buffer system, with [U-14C]palmitate (i.e., the C16 fatty acid, palmitate, labelled with 14C in all 16 carbon atoms), at a specific radioactivity of 103 dpm /mmol, with and without the addition of 60 mmol/L malonate and / or oxaloacetate.
One set of incubations was set up to act as an unincubated control, in which the perchloric acid was added at the beginning of the incubation.
After collection of the carbon dioxide for measurement of radioactivity, the denatured incubation mixture was extracted with a chloroform:methanol mixture to separate unmetabolised fatty acid (palmitate, in the organic phase) from water-soluble metabolites (in the aqueous layer). The radioactivity in both phases was determined.
radioactivity (1000 dpm
/min /g cells) in: |
|||
carbon dioxide |
organic phase |
aqueous phase |
|
| unincubated control | 0 |
10.0 |
0 |
| no addition | 2.3 |
7.5 |
0.2 |
| + malonate (an inhibitor of succinate dehydrogenase) |
0 |
9.8 |
0.2 |
| + oxaloacetate | 4.8 |
5.0 |
0.2 |
| + oxaloacetate + malonate | 0 |
5.0 |
5.0 |
What conclusions can you draw from these results?