Ammonia Metabolism Lecture Sep 4

Question 1

What is the nitrogen balance?

Answer 1

Nitrogen balance [grams] = nitrogen intake [g] – urinary urea nitrogen [g] – X [g]

Question 2

WHy is it important that we have a lot of excess capacity in the urea cycle?

Answer 2

So we have the capcity to remove all the nitrogen we take in–regardless of how much it is.

Question 3

What does it mean to be in nitrogen balance?

What it is in health adults?

WHat is it in children?

What is it in people with anorexia, kwarshiorkor, or chachexis?

Answer 3

Healthy adults will excrete as much nitrogen as they consume.

Children (or anyone who is trying to greatly increase their mass) will have a positive nitgrogen balance–they do not excrete all the nitrogen they consume.

Anorexic individuals will have a negative nitrogen balance because they break down muscle for energy, and the nitrogen from the muscle breakdown is excreted in excess of the nitrogen they take in.

Question 4

What is the ratio of nitrogen to protein in the protein we eat?

Answer 4

We use the constante 0.16 g N/1 g protein

Question 5

How is most of the nitrogen excreted in urine? What are some other ways?

Answer 5

90% excreted at urea

Some extreated as NH4+, creatinine, and uric acid

Question 6

How do amino acids get from the food we eat to our blood?

In other words, how are amino acids digested?

Answer 6

1. THe proteins are denatured by the low pH in the stomach.
2. Pepsin is a protease that is secreted in the stomach, which will cleave other proteases in the gut to make them active, including trypsinogen, chymotrypsinogen, etc.
3. The proteases in the intestinal lumen will break down proteins to singel dr- and tri-peptides.
4. These single di- and tri-peptides are then taken up by the gut epithelial cells.
5. WIthin the epithelial cell, the di and tri-peptides will be further broken down into single AAs.
6. The AAs then pass through the basal lateral membrane of the gut epithelial cells into the blood circulation.

Question 7

Under what conditions does nitrogen need to be put on urea for excretion?

Answer 7

WHen there is an excess of AAs in the blood (like after eating a high protein meal)

During prolonged starvation when the body is having to use AAs for fuel.

Question 8

Where is urea synthesized?

Answer 8

In the liver

Question 9

There usually isn’t a lot of free nitrogen in the blood, but what little is in the blood comes in the form of ammonia and ammonium

What is the relationhsip between ammonium ion (NH4+) and ammonia (NH3)?

How does pH affect the balance?

Which one can pass through membranes and which one can’t?

Answer 9

In a solution with a pH of 9.3, there will be equal concentrations of ammonium ion and ammonia.

So at the body’s pH level, the ammonium ion (NH4+) will predominate.

This is significant because ammonium ion (NH4+) can’t cross membranes, while ammonia (NH3) can.

Question 10

Where do AAs in the blood go initially?

Answer 10

To the liver

Question 11

How are amino acids transported from the gut lumen into the intestinal epithelial cells?

How are they transported from the epithelial cells into the blood?

Answer 11

They use secondary active transport, coupled with sodium’s transport down its gradient through a symport.

This is possible due to proton pumps that pump Na out of the cell using ATP hydrolysis.

To get into the blood from the gut epithelial cells, all it takes is facilitated diffusion trhoguh a transporter.

Question 12

What are the 5 key amino acids we need to remember for protein digestion and amino acid turnover?

What are their associated keto-acids?

Answer 12

Glutamate: alpha ketoglutarate

Aspartate: oxaloacetate

Alanine: pyruvate

Glutamine: Glutamate (:alpha ketoglutarate)

Question 13

What is glutamate’s importance in amino acid digestion and nitrogen balance?

Answer 13

Glutamate basically makes up the amino group pool of the cell.

If a cell needs to synthesized AAs, it makes the carbon skeleton and then takes the nitrgoen from glutamate in a transaminase reaction resulting in formation of the new AA and alpha ketoglutarate.

If the cell wants to break down AAs, it takes the nitrogen from the amino acid and puts it on alpha ketoglutarate to make glutamate

Question 14

What is the importance of aspartate in protein digestion/AA turnover?

Answer 14

Aspartate is the AA that donates nitrogen to the urea cycle.

So any nitrogen that isn’t free before it enters the urea cycle is on aspartate.

Question 15

What is the importance of alanine in protein digestion/AA turnover?

Answer 15

Alanine/pyruvate play an important role in gluconeogenesis.

In the fasted state, if a muscle cell is doing work it will break down some of its protein and all of the amino acids will be liberated. Although the branched AAs will be used for energy right in that muscle cell, other AAs particularly alanine will go to the liver, where it will then be added to a carbon skeleton to make glucose in gluconeogenesis.

Question 16

What is the role of glutamine in protein digestion and amino acid turnover/urea cycle?

Answer 16

Glutamine is the primary way nitrogen is transported to the liver for the urea cycle.

Question 17

In the FED state, what is the fate of AAs entering the circulating pool of AAs in the blood?

In other words, what effect does insulin have on protein synthesis?

Answer 17

Since in the fed state there is abundant fats and carbs for energy production, the AAs are stored.

THe primarily storage for AAs is through protein.

Therefore, insulin promotes storage pathways, including translation/synthesis of proteins.

Question 18

Even though most AAs in the fed state are stored in the form of proteins, there are 2 other ways the AAs can be used for storage. What is this?

Answer 18

The AAs can be deaminated and then metabolized down to acetyl CoA, which can be used to make TGs, which can then be transported out of the liver in VLDL to be stored in adiose tissue.

They can also be deaminated and the carbon skeleton used to make glucose (for RBCs in the event of a high protein meal) and then glycogen for storage of carbs.

Question 19

During the fasted state, AAs entering the AA pool in the blood won’t be coming from the dietary AA in the gut, they’ll be coming from proetin breakdown in muscle.

Branched amino acids will be used in the muscles through the TCA cycle for energy.

What two amino acids are the primary nitrogen carriers that leave the cell in this situraiton?

Where do they each go after leaving the muscle?

WHere do they both end up?

Answer 19

Glutamine and alanine.

Glutamine can be used for fuel in a variety of tissues including the kidney and gut. As these are used, the glutamine is converted to alanine. (and the nitrogen broken off in the liver is excreted as urine)

Alanine is transported to the liver to be used for gluconeogensis (to provide fuel for the brain and RBCs)

Then everything goes to the liver where they can be used in gluconeogenesis to provide glucose, ketogenesis to provide fuel for the muscle, kidney, and eventually the brain. THe nitrogen that is taken off these amino acids is fixed to urea, which is then excreted in the urine.

Question 20

What 4 hormones promote the mobilization of stored fuels?

Answer 20

Glucagon

cortisol

epinephrine

norepinephrine

Question 21

Describe the alanine/glucose cycle.

Why is this cycle important?

What is the universal amin acceptor in the cell?

WHat AA eventually trasnports the nitrogen out in this case?

Answer 21

This cycle is important because it allows amino acids to be used as fuel by muscle cells.

In the cycle, proteins are broken down, resulting in amino acids.

To use this as fuel, they need to be deaminated to form alpha keto acids that can enter the TCA cycle as intermediates.

alpha ketoglutarate is the universal amin accepor, takes the nitrogen, and forms glutamate.

Glutamate then donates the N to pyruvate, forming Alanine, which is what carries the nitrogen to the liver.

Question 22

WHat happens to the alanine once it reaches the liver?

Answer 22

Its nitrogen is removed and placed on urea for excretion and its carbon skeleton is used for gluconeogensis.

Question 23

In the muscle cell and peripheral tissues, what 2 enzymes work to essentially “fix” free ammonium?

Answer 23

Deamination of some amino acids including Thr, Ser, His, Glu, Asp, and Gln will result in free ammonium in the cell. Purine nucleotide metabolism will also yield free ammonium in the cell.

This free ammonium is fixed onto glutamine through two enzymes: Glutamate Dehydrogenase (GDH) and Glutamine synthase. Both of these steps require outside energy, first from NADPH (or NAD+ depending on concentration) and then ATP hydrolysis.

Question 24

What happens to the glutamine that leaves the blood and then enters the liver?

Answer 24

The enzyme glutaminase removes the first NH4+ yileding glutamate. THen Glutamate dehydrogenase removes the other NH4+, yielding alpha-KG in the liver.

The two removed NH4+ it added to urea which is excreted.

Question 25

What is special about the glutamate dehydrogenase enzyme? 2 reasons…

Answer 25

1. It can use either NAD+/NADH or NADP+/NADPH as the electron acceptor/donor depending on concentration of the cofactors
2. THe same enzyme is used to add an ammonium to alpha ketoglutarate to yield glutamate in the organs that removes the ammonium from glutamate to yield alpha ketoglutarate in the liver. So it’s reversible and goes in difference directions depending on the organ its on.

Question 26

WHat is the overal function of the urea cycle?

How can nitrogen enter the urea cycle?

Answer 26

It’s to convert nitrogen in the body to urea, which can be excreted in the urine.

Nitrogen enters the cycle as free ammonium and as aspartate.

Question 27

What are the steps in the urea cycle?

Answer 27

Inside the mitochondria:

1. Bicarbonate ion and ammonium ion are combined and phosphorylated through carbamoyl phosphate synthase 1 (CPS1) to yield carbamoyl phosphate
2. THe enzyme ornithine transcarbamoylase binds the carbamoyl phosphate onto ornithine to yield citrulline.
3. Citrulline is transported out of the mitochondria through an antiporter that bring ornithine in and citrulline out.
4. In the cytosol, citrullin is a substrate for arginosuccinate synthase which combines cirtulline (carrying nitrogen that was free on ammonium ion) to aspartate (which is carrying the nitrogen from transaminase reactions within the cell), giving arginosuccinate as a product
5. Arginosuccinate lyase then removes the carbon skeleton of aspartate as fumarate and give us arginine.
6. arginine is the substrate for arginase, which femoves the the HHN-CO–NHH urea, with the leftover being regenerated ornithine
7. THe urea is excreted out and ornithine is brought back into the mitochondria through the antiporter.

Question 28

What is the main regulator of the urea cycle flux?

What enzyme in the urea cycle is allosterically regulated?

Answer 28

THe main regulator of the urea cycle is actually just the concentration of substrates–feedward regulation.

CPS-1 is allosterically regulated

Question 29

How does arginine act as an activator of the urea cycle?

2 ways

Answer 29

The first way is the simpler of the two: it just acts as a feedforward substrate for arginase (convertin arginine to ornithine)

THe second way is through indirect allosteric activation:

Aringine increases synthesis of N-acetyl-glutamate (NAG) by N-acetyl-glutamate synthase. (from glutamate and acetyl CoA)

NAG then acts as an allosteric activator of CPS-1.

Thus, when arginine levels are high, more NAG is synthesized, which activates CPS-1 to increase the urea cycle activity.

This is good because a buildup of arginine suggests the cycle isn’t working fast enough to handle the nitrogen in the body.

Question 30

All urea cycle disorders manifest in what two ways?

Answer 30

hyperammonaemia

hyperglutaminaemia

(high ammonia, high glutamine)

Question 31

WHat are some symptoms of urea cycle disorders?

Why are most of them neurological?

Answer 31

Refusal to eat/protein aversion

seizures

irritabiliy

lethargy

ataxia

tremors

FTT

Most are neurological because when everything is going to glutamine, glutamate is depleted. Unfortunately, glutamate is necessary for neurotransmitter synthesis, so you get neurological symptoms.

Question 32

What is the most common way a patient can develop an issue with the urea cycle?

Answer 32

Liver damage

Question 33

As far as inherited disorders of the urea cycle, what are the 7 targets?

Answer 33

1. NAG synthetase
2. CPS1
3. Ornithin transcarbamoylase
4. Ornithin CItrulline transporter
5. Arginosuccinate synthetase
6. arginosuccinate lyase
7. arginase

Question 34

An error just after the CPS1 reaction (such as with ornithin transcarbamoylase-OTC) will cause a buildup of what molecule in the urine?

Answer 34

You would think it would just cause a buildup of carbamoyl phosphate

BUT carbamoyl phosphate is also used in pyrmidine synthesis, so when there is a buildup of carbamoyl phosphate, it dumps out into the pyrmimidine synthesis pathway and it’s an intermediate further down this pathway that actually builds up in the urine:

orotic acid

Question 35

A patient who has a defect in the ornithine citrulline antiporter, what will their blood work show?

What is this syndrome called?

Answer 35

They have HHH syndrome

Hyperammonaemia (nondescript)

hyperornithaemia (buildup of ornithine because it can’t enter the mitochondria to be combined with carbamoyl phospahte to yield citrullin)

Homocitrullinaemia (a buildup of homocitrulline. This occurs because the carbamoyl phosphate builds up and reacts with lysine (instead of the ornithine it wants since it isn’t there) and forms homocitrulline)

Question 36

How does the lab measure free ammonia in a patient’s blood?

How does a BUN take that one step forward?

(blood urea nitrogen)

Answer 36

Take the blood and incubate it with recominant GDH, alpha ketoglutarate and NADPH.

It there is a high concentration in ammonium ion, more NADPH will be reduces to NADP, which can be measured by UV spectroscopy

A BUN measures the TOTAL ammonia in the blood (what’s free as ammonium ion and what’s fixed as urea)

There’s an additional step before the GDH addition. You add urease first to remove the NH4+ from urea. THen you do the GDH reaction and measure again.

Thus, the BUN will always be higher than the free ammonium measurement alone.

Question 37

What is the order of steps you should take in diagnosing a child with a suspected urea cycle disorder?

Answer 37

1, Obtain a blood pH and bicarbonate measurement (this checks for acidosis)

2. If the patient doesn’t have acidosis, then you should check the patient’s BUN
3. Obtain plasma amino acid counts
4. If there are specific amino acid counts that are elevated, you should can make a definitive diagnosis to a defect in a certain enzyme (based on which enzyme’s substrates are increased and products decreased).
5. If there is no specific amino acid elevation, that issue is probably further up the pathway, so you should check a urine orotic acid
6. If urine orotic acid is low, you know the issue is either in CPS1 or in NAG synthetase. If orotic acid is high, you know it’s after CPS1, so it must be OTC deficiency.

Question 38

What is the overall goal of treatment in urea cycle diseases?

Answer 38

Decrease the concentration of ammonium ion in the blood.

Question 39

What are 5 ways you can reduces the concentration of ammonium ion in the blood?

Answer 39

1. low protein diet
2. Treat with N-carbamoylglutamic acid IF the issue is in NAG snthetase–it acts as an analog of NAG that will allosterically activate CPS-1 and the problem is solved
3. Eliminate nitrogen through other pathways with drugs: arginine, benzoic acid, or phenylbutyrate
4. Liver transplate or hepatocyte transfusion if the issue is due to liver damage
5. Viral transduced gene therapy–infect the patient with a virus vector that has the appropriate gene for the enzyme they’re missing.

Question 40

How do phenylbutyrate and benzoic acid work as treatment for urea cycle disorders?

Answer 40

They are used to make amino acids excretable in the urine so the nitrogen can essentially bypass the urea cycle that isn’t working.

Phenylbutyrate makes bind to glutamine and makes it soluble for excretion in the urine.

Benzoat binds to glycine and makes is soluble as hippuric acid in the urine.

Question 41

Why is supplementing arginine useful in the treatment of urea cycle disorders?

Answer 41

In disorders where arginosuccinate lyase is the issue, you don’t get the conversion of argininosuccinate to arginine, thus you don’t get regeneration of ornithine and the urea cycle blocks up.

If you supplement arginine, you can still have the regeneration of ornithine and ammonia will be pass through the urea cycle and be excreted on argininosuccinate (which is soluble enough) in the urine

.

Question 42

How does treatment with N-carbamoyl-glutamate work against urea cycle disorders? Which disorder does it have to be?

Answer 42

It is used in treatment of defects in NAG synthetase.

If you don’t get synthesis of NAG, CPS-1 isn’t activated and you don’t get production of carbamoyl phosphate.

Thus, N-carbamoyl-glutamate is an analog of NAG and can act in its place as the allosteric activator of CPS-1.

Of course, this won’t work if you don’t have any functional CPS-1 either. Then you have to rely on phenylbutyrate, benzoic acid and a low protein diet.