Ammonia Metabolism

Question 1

Do we use nitrogen for energy?

Answer 1

No. We pee it out as the urea cyle has plenty of excess capacity

Question 2

True, false - Adults have generally run at a nitrogen surpluss, while kids are balanced.

Name a case when adults run at a deficit

Answer 2

False - adults’ nitrogen intake is balanced with nitrogen elimination (except with hardcore bodybuilders)

Kids consume more than they eliminate; have a positive balance.

In anhorexia, adults pee more than they consume

Question 3

Nitrogen balance =

How many grams of nitrogen per gram of protein?

Answer 3

Nitrogen in diet (g) - urinary urea nitrogen (g) - X o

X is a constant ranging from 2-4 based on how food nutrients are taken (oral vs parental). X is higher in kids.

1 gram of protein –> .16 g N

Question 4

Dietary protein intake

Answer 4

Dietary protein is digested down to single amino acids in the gut and intestinal epithelial cells.

Once amino acids enter the blood; they are first transported to the liver.

Question 5

How are amino acids taken up in the intestinal lumen?

Answer 5

Similar to the intestinal SGLT glucose transport systemTransport of amino acids from the gut lumen into intestinal epithelial cells is by secondary active transport.

Question 6

NEED-TO-KNOW AA’s, conjugates and function

Answer 6

Glutamate, a-ketoglutarate - The amino group pool of the cell

Aspartate, oxaloacetate - Donates nitrogen to the urea cycle

Alanine, pyruvate - A key role in gluconeogenesis

Glutamine, glutamate - Transports nitrogen to liver for urea cycle

Question 7

When cells need to breakdown proteins for energy, which ones are used?

Answer 7

Branch-chained AA’s are used directly by cells they’re in. AA’s like Ala are not used for E in their respective cells; rather, they are lopped off, enter the blood stream and head to the liver for gluconeogenesis.

Question 8

Protein digestion and AA turnover. What promotes storage pathways?

Answer 8

Insulin promotes storage pathways b/c indicates glucose is around, which is preferred.

Question 9

During the fed state, dietary AA’s enter which pool? The Protein pool for storage or the AA pool for energy generation?

Answer 9

AA’s enter the protein pool via translation during fed states. AA’s enter the AA pool via proteolysis during fasting, but more over starved states.

Question 10

If branch chain AA’s are used by muscle cells for energy during fasting states, glutamine and alanine –> liver for gluconeogenesis in liver –> glucose. What are circulating hormones that regulate this?

Answer 10

Glucagon, cortisol, epinephrine and norepinephrine promote mobilization of stored fuels.

Question 11

Protein digestion during fasting

Answer 11

Question 12

In fasted state,_____ promotes protein breakdown. _______ is the main intracellular pool of amino groups; it donates N, while it’s conjugate (alpha-KG) accepts N groups to/from other AAs. What drives this?

Answer 12

Cortisol –> protein breakdown during fasted states.

Glutamate = main intracellular pool of amino groups.

Glutamate/alpha-KG ratio is concentration-dependent; no ATP req’d

Question 13

________ and _______ can add free ammonium to “fix” it as glutamine. These reactions require energy. True/false - this is a transamination.

Is E gained from breaking another AA bond?

Answer 13

Glutamate dehydrogenase and glutamine synthase.

False- GDH and glut sythase fix “free” ammonium derived from deamination of other AA’s

E does NOT come from breaking another AA bond. Rather, it comes from ATP hydrolysis

Question 14

What 3 enzymes fix free nitrogen (NH4+/NH3)?

Answer 14

Glutamante dehydrogenase, glutamine synthetase and carbamoyl phosphate synthetase

Question 15

Glutamate dehydrogenase

Answer 15

1. One of three enzymes that can fix free ammonium.
2. Reversible.
3. Can use either NAD+/NADH or NADP+/NADPH as the electron acceptor/donor

Question 16

What is the function of the Urea Cycle?

Answer 16

Function is to convert nitrogen to urea, which can be excreted in the urine.

Question 17

What two ways does nitrogen enter the urea cycle?

Answer 17

As free ammonium and as aspartate

Question 18

What is ornithine in the Urea cycle analagous to?

Answer 18

Ornithine plays an analogous role as oxaloacetate in the TCA cycle.

Question 19

Urea cycle overview

Answer 19

Question 20

_____ is a key allosteric regulator of the urea cycle.

Answer 20

ARGENINE increases synthesis of N-acetyl-glutamate (NAG) by N-acetyl-glutamate synthase. NAG acts as an allosteric activator of carbamoyl phosphate synthetase I.

It’s the substrate that contains urea. After argenase lopps off urea, ornithine is regenerated and heads back into the mitochondria to join carbamoyl phosphate to form citrulline.

Question 21

What is the main regulator of the urea cycle?

Answer 21

The liver has a high capacity to fix nitrogen as urea. The availability of substrates is the main regulator of urea cycle flux (feed forward regulation).

Question 22

How else is the urea cycle regulated?

Answer 22

The urea cycle is also modulated by allosteric regulation of CPS-I and transcriptional regulation of urea cycle enzymes.

Question 23

What does lots of argenine in BS mean?

Answer 23

Lots of arginine suggests backup of urea cycle. Drug target if inherited defect in N-acetyl-glutamate synthase.

Question 24

Argenine impacts urea cycle how?

Answer 24

Question 25

What two ways does argenine activate the urea cycle?

Answer 25

1. When arginine builds up. . .
   It increases the synthesis of N-acetyl-glutamate (NAG). NAG allosterically activates CPS-I.
2. It increases arginase activity

Question 26

How do urea cycle dissorders usually manifest?

Answer 26

hyperammonaemia and hyperglutaminaemia

Question 27

Neurons are particularly sensitive to ammonium toxicity. Glutamate depletion prevents synthesis of neurotransmitters. Symptoms include:

Answer 27

Refusal to eat / protein aversion
Seizures
Irritability
Lethargy
Ataxia
Tremors
FTT

Question 28

Six enzymes, six inherited disorders of the urea cycle. All manifest as elevated blood [NH4+].

Answer 28

Question 29

CPTII

Answer 29

Carbamoyl phosphate is also a substrate for pyrimidine synthesis. Defects close to CPTII –> elevated levels of urniary orotic acid

Question 30

What affect would knocked out CPTI have on urinary orotic acid levels?

Answer 30

Defect in CPM 1 would probably –> decreased orotic acid levels b/c it precedes carbamoyl phosphate

Elevated urinary orotic acid is characteristic of urea cycle disorders downstream of CPS-I

Question 31

Inherited HHH Disorders: What is elevated and what is defective?

Answer 31

In hyperammonaemia, hyperornithaemia, homocitrullinaemia syndrome (HHH syndrome), the ornithine / citrulline antiporter is defective.

Question 32

How to diagnose inherited urea-cycle disorders in patients?

Answer 32

Measure free ammonium in blood - incubate their blood with recombinant GDH, a-ketoglutarate, and NADPH (reduced). NADPH absorbs UV light, so absorption decreases as it is oxidized. The decrease in light absorption is proportional to the concentration of NH4+.

BUN (blood urea nitrogen) - To measure BUN, the patient’s sample is first incubated with bacterial urease, then the GDH reaction is performed

Question 33

Clinical approach to a newborn with hyperamonaemia

Answer 33

Question 34

Goal of Tx in patients w/ hyperammonaemia

Answer 34

The goal of treatment: Decrease blood [NH4+]
*Low protein diet

*N-carbamoylglutamic acid – acts as allosteric activator of CPSI

*Eliminate nitrogen in alternative pathways: to increase excretability of NH3
Arginine
Benzoic acid
Phenylbutyrate

*Liver transplant / hepatocyte transfusion

*Viral transduced gene therapy – promising, but very risky

Question 35

________ and _______ are used to make amino acids excretable in the urine

Answer 35

Phenylbutyrate and benzoic acid.

• Phenylbutyrate + glutamine (important N transporter AA) crx highly soluble/excretable compounds
• Benzoate bipasses urea cycle altogether

Question 36

What can be used in the case of arginosuccinate lyase deficiency?

What gets excreted?

Answer 36

ARGENINE

Dumping of arginine activates urea cycle, but arginosuccinate is what’s excreted