1
MB ChB PHASE I
HOW THE BODY
HANDLES NITROGEN
LECTURE 1
AIM: To review:
N-containing molecules of the body;
N flow through the biosphere;
N flow through the body;
digestion of dietary protein;
role of glutamate in transfer of N to,
from and between amino-acids.
[C&H Chapters 19, 20; pp.367-369]
http://www.abdn.ac.uk/~bch118/index.htm
2
N-CONTAINING
THE BODY
MOLECULES
OF
The main ones are:
1 Proteins (contain amino-acids)
2 Nucleic acids and nucleotides
(contain bases)
3 Active amines
CO2
amino-acid
1
/2O2
+
NH4
amine
aldehyde
ACTIVE
INACTIVE
(several,
non-coded)
INACTIVE
3
eg
histamine
serotonin
dopamine
(nor-)epinephrine.
4 Haem-containing
cytochromes.
haemoglobin
and
(Proteins and Terminal Respiration lectures)
4
N FLOW THROUGH THE BIOSPHERE
N in biomolecules comes ultimately from
N2 (80% of the atmosphere).
However, N2 isn’t readily exchanged with
N found in organisms.
(Compare with ready exchange
environmental and biological C, H, O.)
between
This is because N2 is very unreactive:
N2
is
N
N.
Only a few micro-organisms can reduce N2
and ‘fix’ it in the biosphere.
Once ‘fixed’, as NH4+, N flows to other
biomolecules through the (coded) aminoacid glutamate.
[For structure, see Proteins Lecture 2.]
Only a few (different) micro-organisms
can pass N back to the atmosphere, as N2.
5
ATMOSPHERE
N2
BIOSPHERE
+
NH4
glutamate
(other) amino-acids
proteins and other N-containing
biomolecules
amino-acids
glutamate
+
NH4
N2
6
N FLOW THROUGH THE BODY
The main flow involves:
1
digestion of dietary protein;
‘turn-over’ of body protein;
excretion of N as urea;
2
a pool of intra- and extra-cellular
free amino-acids
(kept constant at about 100g).
7
body
protein
dietary
protein
synthesis of
some
(‘non-essential’)
amino-acids
amino-acid
pool
body
protein
synthesis of
other
N-containing
molecules
N excreted
as urea
C
or
ATP
glucose (gluconeogenesis)
fatty acids
ketone bodies
CO2
8
DIETARY PROTEIN
Dietary protein replenishes amino-acids used in
anabolism and catabolism.
(as shown in the previous diagram)
Normally, N flow into and out of the body is
balanced.
N flowin that is less than N flowout results in
‘negative nitrogen balance’.
This happens in the condition kwashiorkor.
[‘Sickness of the older child when the next baby is born’]
It occurs when carbohydrate intake is adequate,
but N intake is poor,
as sometimes happens when a child is weaned
onto a starchy diet.
9
Among several consequences:
low N intake
low plasma albumin
low plasma osmotic pressure
lower than usual entry of interstitial water into plasma
oedema
plump belly
N flowin that is greater than N flowout results in
‘positive nitrogen balance’.
This occurs during rapid tissue growth:
eg in children;
pregnancy;
recovery from illness;
body-building.
N taken in in excess of requirements
cannot be stored (unlike carbohydrate, fat)
and is excreted.
10
Of the 20 coded amino-acids,
10 (‘non-essential’) can be synthesised in the
body (they have simple R groups);
8 (‘essential’) cannot be synthesised and must
be present in the diet;
2 (arginine, histidine) can usually be made at
adequate rates, but are needed in the diet
when tissue growth is rapid.
Animal protein is usually rich in all essential
amino-acids.
An exception is collagen (in gelatin).
[Proteins Lecture 4]
Particular plant proteins are deficient in some
essential amino-acids.
Vegetarians need to use a mix of protein
sources.
11
DIGESTION OF DIETARY PROTEIN
1
Stomach
HCl denatures proteins and makes them
accessible to degradative enzymes.
The zymogen pepsinogen is cleaved to pepsin
autocatalytically, and, later, by pepsin itself.
Pepsin cleaves proteins to small polypeptides.
2 Small intestine
Mucosal cell-surface enteropeptidase cleaves
trypsinogen (secreted by the pancreas) to
trypsin.
Trypsin cleaves other pancreatic zymogens to
elastase
[met lung elastase in Proteins Lecture 4];
chymotrypsin;
carboxypeptidases A and B.
12
These enzymes have different specificities:
they cleave adjacent to different amino-acids.
Together, they break polypeptides to free
amino-acids and short peptides.
In
addition,
a
mucosal
cell-surface
aminopeptidase removes amino-acids one at a
time from N termini.
Amino-acids and short peptides are absorbed
into mucosal cells by several methods,
including
ATP-driven
Na+-dependent
transport like that used for Glc.
[Energy Transformations - Carbohydrates Lecture 2]
Absorbed peptides are broken to amino-acids
by a cytosolic peptidase.
Amino-acids move through the portal system
to the liver and are either metabolised directly
or released into the general circulation.
13
body
protein
dietary
protein
synthesis of
some
(‘non-essential’)
amino-acids
amino-acid
pool
body
protein
synthesis of
other
N-containing
molecules
N excreted
as urea
C
or
ATP
glucose (gluconeogenesis)
fatty acids
ketone bodies
CO2
14
ROLE OF GLUTAMATE IN TRANSFER OF
N TO, FROM AND BETWEEN AMINOACIDS
Glutamate (Glu) is one of the 20 coded
amino-acids.
(Proteins Lecture 2]
It forms a link in the flow of N
between +NH4 and other amino-acids
(see earlier ‘Biosphere’ diagram),
and from one amino-acid to another.
It does this by taking part in 2 reactions:
15
1 oxidative deamination
NAD(P)H + H+
+
NH4
+
-ketoglutarate
NAD(P)+
Glu
+
(KG)
COO-
COO-
(CH2)2
(CH2)2
C O
COO-
HC
NH2
COO-
KG is a citric acid cycle intermediate.
[Terminal Respiration Lecture 1]
H2O
16
The reaction:
can use either NAD or NADP;
can proceed in either direction (depending on
the conditions);
is an oxidative deamination when it proceeds
R
L;
is catalysed by
(named for the R
glutamate dehydrogenase
L reaction);
is crucial to life:
Glu is the ONLY amino-acid that can obtain its
N directly from +NH4
(in the L
R reaction).
17
All others obtain their N from a pre-existing
amino-acid
(as in the following reaction).
2 transamination (aminotransfer)
Glu
eg
+
-ketoacid
pyruvate
CH3
C O
COO-
-KG + amino-acid
alanine
CH3
HC
NH2
COO-
and
oxaloacetate
aspartate
COO-
COO-
CH2
CH2
C O
COO-
HC
NH2
COO-
18
These reactions:
can proceed in either direction (depending on
the conditions);
are catalysed by transaminases
(aminotransferases).
use pyridoxal phosphate
(made from vitamin B6) as a cofactor.
Measurement of these normally intracellular
enzymes in plasma
allows progression of liver and heart disease
(in which cell damage and enzyme leakage
occur)
to be followed.
19
In summary:
Glu, through these two reactions, plays a
central role in:
1 movement of N from +NH4 to amino-acids
+
NH4
KG
amino-acid
Glu
-ketoacid
reaction 1
reaction 2
2 movement of N from amino-acids to +NH4
amino-acid
KG
-ketoacid
Glu
reaction 2
+
NH4
reaction 1
20
3 transfer of N from one amino-acid to
another
amino-acid 1
KG
amino-acid 2
-ketoacid 1
Glu
-ketoacid 2
reaction 2
reaction 2
21
MB ChB PHASE I
HOW THE BODY
HANDLES NITROGEN
LECTURE 2
AIM: To review:
catabolism of body protein;
transport of N to the liver;
formation of urea for excretion.
[C&H Chapters 19, 20; pp.367-369]
22
CATABOLISM OF BODY PROTEIN
This is part of the N flow through the body
that we saw in Lecture 1:
body
protein
dietary
protein
synthesis of
some
(‘non-essential’)
amino-acids
amino-acid
pool
body
protein
synthesis of
other
N-containing
molecules
N excreted
as urea
C
or
glucose (gluconeogenesis)
fatty acids
ketone bodies
ATP
CO2
23
OVERVIEW OF THE FLOW OF N FROM
CATABOLISED PROTEIN TO UREA
body protein
1
amino-acids
2
Glu
3
+
NH4
4
glutamine
transported through plasma to liver
5
+
NH4
+
Glu
6
or
7
aspartate
+
NH4
urea
transported through plasma to kidney for excretion
24
Also, in skeletal muscle:
2
Glu
8
alanine
transported through plasma to liver
9
Glu
10
aspartate
or
+
11
NH4
25
FLOW OF N FROM
PROTEIN TO UREA
CATABOLISED
1 300-400g body protein is ‘turned-over’
daily.
It is broken to amino-acids by intracellular
proteinases.
2 Amino-acid
-amino
group
N
transferred to Glu by transamination.
(as seen in Lecture 1)
amino-acid + KG
N
Glu + -ketoacid
N
3 Glu is oxidatively deaminated.
(as seen in Lecture 1)
Glu
N
KG
+
+
NH4
N
is
26
4 Ammonia is very toxic.
Hyperammonaemia (high plasma [+NH4])
causes
tremors;
speech slurring;
coma;
death.
Probably,
high [+NH4]
pulls the oxidative deamination reaction [Step 3]
towards Glu synthesis
KG, a component of the citric acid cycle, is
depleted
ATP production by oxidative phosphorylation
decreases
brain is vulnerable to low [ATP].
27
Also, perhaps high [+NH4] depletes ATP
by excessive flow through the glutamine
synthetase reaction [see below].
And Glu and its derivative -aminobutyrate
are concerned with neurotransmission:
perhaps changes in their concentrations
when [+NH4] is high also contribute to the
symptoms.
Because of ammonia toxicity, N is
transported from many body tissues
through plasma as non-toxic glutamine
[one of the 20 coded amino-acids].
ATP
+
NH4
N
ADP + Pi
glutamine synthetase
glutamine
2N
Glu
N
COO(CH2)2
CONH2
(CH2)2
28
5 In the liver, glutamine is converted back to
+
NH4 and Glu.
H 2O
+
glutamine
NH4 +
Glu
N
N
glutaminase
2N
6 Glu now either passes its N to aspartate by
transamination (as seen in Lecture 1),
Glu + oxaloacetate
N
KG + aspartate
N
7 or is oxidatively deaminated [as in step 3].
Glu
N
KG
+
+
NH4
N
29
The N of the +NH4 and aspartate formed in the
liver in steps 5-7
becomes N of urea.
Skeletal muscle can also transport N from its
degraded proteins through plasma as alanine.
[See the ‘Overview’ diagram]
8 Glu,
instead
of
being
deaminated [as in step 3],
is transaminated to alanine
oxidatively
[as seen in Lecture 1].
Glu
N
+
pyruvate
KG +
alanine
N
Non-toxic alanine is now transported
through plasma.
30
9 In the liver, alanine is transaminated back
to Glu.
[reverse of step 8]
10 Glu is then either transaminated to
aspartate.
[as in step 6]
11 or oxidatively deaminated to give +NH4.
[as in step 7]
As before, the N of the +NH4 and aspartate
formed in the liver in steps 10 and 11
becomes N of urea.
31
Why transport N as glutamine or alanine,
rather than Glu?
Glu has a net negative charge:
its transport would mean additional transport
of a cation;
also, its charge means it does not readily pass
through membranes.
Alanine and glutamine bear no net charge.
[The reason why alanine in particular is transported
from skeletal muscle is discussed in Lecture 3.]
32
FORMATION OF UREA FOR EXCRETION
N from catabolised protein is now in the form of
aspartate and +NH4 in the liver.
It is transformed into N of urea.
33
The urea cycle is shown in C&H, p. 254.
34
To make it simpler:
2N (as urea)
1N (from +NH4 via
carbamoyl phosphate)
2N
4N
3N
4N
1N (from aspartate)
35
Another version:
[1N] [1C]
+
NH4 + CO2
2ATP
2ADP + Pi
NH2
CO
O
OPO
O
H2N
C O
H2N
urea
carbamoyl
phosphate
[2N, 1C]
ornithine
arginine
citrulline
aspartate
[1N]
fumarate
ATP
AMP + PPi
arginosuccinate
36
Arginine is one of the 20 coded amino-acids;
ornithine and citrulline are uncoded aminoacids.
Carbamoyl
phosphate
and
citrulline
syntheses occur in the mitochondrial matrix;
the other reactions are cytosolic.
Note the energy expended in excreting N as
urea.
Urea passes from the liver through plasma to
the kidney for excretion.
37
Returning to hyperammonaemia:
‘Acquired hyperammonaemia’ may occur
during liver malfunction,
as in cirrhosis caused by
alcoholism;
hepatitis;
bile duct obstruction.
In such conditions,
metabolism of N in the liver is prevented,
and [plasma +NH4] increases.
‘Hereditary hyperammonaemia’ occurs when
one or other of the urea cycle enzymes is
genetically deficient.
38
Treatment includes:
1 lowering (but not eliminating) protein
intake, to reduce load on the urea cycle.
2
iv benzoate
glycine
iv phenylbutyrate
Gln
Glycine and Gln synthesis,
to replenish that removed,
helps reduce [plasma +NH4].
non-toxic,
readily
excreted
products
39
3 Partial deficiency in a urea cycle enzyme
may be treated by specific supplementations
/limitations in diet.
Total deficiency of an enzyme is likely to
be fatal.
Kidney failure can
hyperammonaemia.
also
result
in
A little liver urea passes (quite normally) to
the gut lumen.
Most is broken to +NH4 by bacteria.
Some of this is lost in faeces; some moves into
plasma.
In kidney failure,
more urea than usual passes to the gut,
and more than usual +NH4 produced there
moves into plasma.
Treatment:
oral antibiotics to inhibit bacterial
synthesis.
+
NH4
40
MB ChB PHASE I
HOW THE BODY
HANDLES NITROGEN
LECTURE 3
AIM: To review:
fate of the C of catabolised body protein;
why alanine is transported from muscle;
removal of glutamine by the kidney;
phenylketonuria.
[C&H Chapters 19, 20; pp.367-369]
41
FATE OF THE C OF CATABOLISED
BODY PROTEIN
Refer back to the N flow through the body
that we saw in Lecture 1:
body
protein
dietary
protein
synthesis of
some
(‘non-essential’)
amino-acids
amino-acid
pool
body
protein
synthesis of
other
N-containing
molecules
N excreted
as urea
C
or
glucose (gluconeogenesis)
fatty acids
ketone bodies
ATP
CO2
42
and recollect the opening steps of the flow of
N from catabolised protein to urea that we
saw in Lecture 2.
They were:
body protein
1
amino-acids
2
2
Glu
3
+
NH4
Glu
or, in skeletal muscle,
8
alanine
43
Step 2 was the transamination:
amino-acid + KG
N
Glu + -ketoacid
N
This can also be represented thus:
KG
amino-acid
N and C
Glu
N
-ketoacid
C
We traced the flow of N
(eventually to urea).
What happens to the C?
44
C of amino-acids is converted into the C of
intermediates of:
glycolysis/gluconeogenesis;
citric acid cycle;
lipid metabolism pathways.
Sometimes the conversion is simple:
eg the C of alanine
and aspartate,
by transamination,
becomes the C
of pyruvate
and oxaloacetate
respectively [as we saw in Lecture 1].
For other amino-acids, the conversion
involves several steps.
45
To summarise, the flow for the various C
atoms of the various amino-acids is:
phosphoenol
pyruvate
pyruvate
acetyl CoA
oxaloacetate
acetoacetate
citrate
fumarate
succinylCoA
KG
46
Any of these flows allows C atoms to be
metabolised to CO2
through terminal respiration,
providing ATP by oxidative phosphorylation.
Those C atoms that flow to
pyruvate or citric acid cycle intermediates
[unfilled arrows in the diagram]
can also generate Glc
by gluconeogenesis.
This occurs at a high rate during
starvation,
when glycogen (and triacylglycerol) stores
are depleted,
and muscle protein is degraded to provide
Glc .
[Proteins Lecture 1 (Protein Function 7);
Terminal Respiration Lecture 1]
47
[Remember,
from Terminal Respiration Lecture 1,
when a citric acid cycle intermediate is produced,
as in the scheme above,
the ‘feed-in’ reaction allows a section of the cycle
to become part of a linear pathway,
and contribute to anabolism (here, of Glc).]
In contrast, those C atoms that flow to
acetyl CoA or acetoacetate
[filled arrows in the diagram],
can flow to fatty acids and (other) ketone
bodies,
but are not gluconeogenic,
for the reasons given in
Terminal Respiration Lecture 1:
pyruvate dehydrogenase-catalysed
irreversible;
reaction
is
there is no net oxaloacetate synthesis through the
citric acid cycle.
48
Amino-acids providing
pyruvate or citric acid cycle intermediate
C atoms
are therefore called ‘glucogenic’.
Those providing
acetyl CoA or acetoacetate C atoms
are called ‘ketogenic’.
Some amino-acids
are both glucogenic and ketogenic
(their different C atoms flow to different
places).
49
WHY
DOES
SKELETAL
MUSCLE
TRANSPORT N FROM CATABOLISED
PROTEIN AS ALANINE?
Recollect from Lecture 2 that other tissues
transport the N as glutamine, but that
muscle, in addition, can transport N as
alanine.
most body tissues
working skeletal muscle
body protein
1
amino-acids
2
2
Glu
Glu
3
+
8
NH4
alanine
4
glutamine
transported through plasma to liver
50
Step 8 in the scheme above is the
transamination:
Glu
N
+
KG +
pyruvate
alanine
N
We traced the flow of the N
(eventually to urea).
But the alanine also carries C from the
skeletal muscle to the liver.
The C comes
pyruvate.
from
skeletal
muscle
[see reaction above]
In the liver, the transported alanine is
transaminated back to Glu and pyruvate.
[Step 9 in Lecture 2]
51
Thus, pyruvate,
generated by glycolysis
in working skeletal muscle,
is transported to liver
(in the form of alanine),
where it can be used in gluconeogenesis.
This process
is similar to, and backs up,
the Cori cycle,
in which lactate
is moved from working skeletal muscle
to the liver.
[Energy Transformations - Carbohydrates Lecture 4]
Thus alanine transport
simultaneously provides
safe movement of N from catabolised muscle
protein,
and
movement of pyruvate C for regeneration of
Glc.
52
REMOVAL OF GLUTAMINE BY THE
KIDNEY
We have seen that N travels from many
tissues through plasma as glutamine,
and that the N of the glutamine, in liver,
eventually becomes N of urea.
Glutamine is also taken up by kidney, where,
through glutaminase and glutamate
dehydrogenase activity
(Steps 5 and 7 of the scheme in Lecture 2],
it is converted to 2 +NH4 and KG.
This usually minor process increases
during metabolic acidosis.
[Acid-Base Balance lectures to come]
Removal of protons as +NH4 in the urine
helps increase plasma pH,
as does KG metabolism through the citric
acid cycle,
because it generates CO2 (bicarbonate).
53
PHENYLKETONURIA
Normally,
phenylalanine (Phe)
(one of the 20 coded amino-acids),
is hydroxylated to tyrosine
(another of the 20).
[See R groups in Proteins Lecture 2]
A genetic defect in the enzyme responsible
leads to increased
[Phe and its metabolites]
in plasma and urine.
Untreated,
this leads to mental retardation,
perhaps because the high [Phe]
competes with movement of other aminoacids across the blood-brain barrier.
Neonates are routinely screened for plasma
Phe.
54
Treatment:
restriction of dietary Phe
(it is one of the ‘essential’ amino-acids)
[Lecture 1]
to amounts
required for protein synthesis.
Foods containing the artificial sweetener
aspartame,
which contains a Phe derivative,
carry a health warning
for those on a Phe-controlled diet.