Metabolism
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Urea synthesis in the liver, and potentially fatal hyperammonaemia in a child
Studies with isolated hepatocytes
Krebs
and Henseleit used very thin slices of liver, approximately one cell thick,
for their studies. The results here are from more recent experiments using isolated
liver parenchymal cells (hepatocytes).
Isolated 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.
The main urinary
nitrogen metabolite in mammals is urea.
What is the main factor that determines how much urea is synthesised and excreted?
The dietary intake of protein is the main factor that determines how much urea is synthesised and secreted, since protein is the main source of nitrogenous compounds in the diet. Remember that for an adult in nitrogen balance the excretion of nitrogenous compounds (mainly urea in the urine) is equal to the dietary intake of nitrogenous compounds. See the exercise on Nitrogen balance and protein requirements for more on this topic.
When isolated hepatocytes with increasing concentrations of ammonium, there is a steady increase in the formation of urea at low concentrations of ammonium, levelling off as the pathway for urea formation becomes saturated. If such studies are performed with isotopically labelled ammonium (15N, a stable isotope) only one of the two N atoms in urea is labelled.
The
sources of ammonium ions in the liver
There are two ways in which ammonium is formed in the liver: from glutamine by the action of glutaminase, and from adenosine, by the action of adenosine deaminase. Each provides about half the ammonium that is incorporated into urea directly.
Glutamine is formed from ammonium in peripheral tissues, as a way of transporting ammonium arising from amino acid and amine metabolism to the liver.
What is the pathway of glutamine formation in peripheral tissues?
Glutamine is synthesised by incorporation of two mol of ammonium into alpha-ketoglutarate, forming first glutamate, then glutamine.
In the liver, glutamine is formed from glutamate and ammonium in the perivenous cells to prevent the escape of ammonium into the peripheral circulation.
See the exercise on Hyperammonaemic coma due to liver failure for the importance of glutamate dehydrogenase in ammonia metabolism, especially in the brain.
The reaction of adenosine deaminase, shown in the lower half of the diagram on the right, and the reamination of inosine to adenosine at the expense of aspartate, occur in the liver, and provide a pathway for the formation of ammonium from aspartate.
Experiment
1: the effect of arginine on urea synthesis
One possible source of urea is the reaction of arginase, which catalyses hydrolysis of the amino acid arginine to yield urea and ornithine, as shown on the right.
Isolated hepatocytes were incubated with varying concentrations of ammonium chloride and 0, 2.5, 5 or 10 mmol /L arginine. The reaction was stopped after 30 min by addition of trichloroacetic acid, and denatured protein was removed by centrifugation. The amount of urea in the supernatant form each incubation was measured by reaction with diacetyl monoxime, ferric ions and thiosemicarbazide to form a red colour that was measured at 540nm.
The results
are shown in the table below and the graphs on the right.
Urea formed (mmol /L) in the presence of different concentrations of arginine, with no added ammonium or a saturating amount (100 mmol /L)
arginine added (mmol /L) |
||||
| ammonium added (mmol /L) | 0 |
2.5 |
5 |
10 |
0 |
0 |
2.5 |
5.0 |
9.9 |
100 |
5.6 |
23.0 |
40.8 |
73.0 |
What conclusions can you draw from these results?
When no ammonium is provided to the hepatocytes, there is stoichiometric formation of urea from arginine. 1 mol of urea is formed fro each mol of arginine added.
However, in the presence of a saturating amount of ammonium there is considerably more urea formed per mol of arginine than can be accounted for by the amount of arginine added.
This suggests that arginine either has a catalytic effect or acts in some other way to increase the rate of formation of urea from ammonium.
Experiment 2: The effect of ornithine on urea synthesis
Since the product of the reaction catalysed by arginase is ornithine, it will be interesting to see the effect of adding ornithine to the incubations. In this experiment, isolated hepatocytes were incubated with varying concentrations of ammonium chloride and 0, 2.5, 5 or 10 mmol /L ornithine, for 30 minutes. The reaction was stopped and the amount of urea formed was measured as described for experiment 1.
The results
are shown in the table below and the graphs on the right.
Urea formed (mmol /L) in the presence of different concentrations of ornithine, with no added ammonium or a saturating amount (100 mmol /L)
ornithine added (mmol /L) |
||||
| ammonium added (mmol /L) | 0 |
2.5 |
5 |
10 |
0 |
0 |
0 |
0 |
0 |
100 |
5.9 |
14.1 |
27.3 |
54.0 |
What conclusions can you draw from these results?