Protein Digestion, Nitrogen Removal from Amino Acids, Transport of NH3 to Liver, Urea Cycle, NH3 Metabolism, Hyperammonemia

1. NITROGEN DISPOSAL
Nitrogen disposal, ch 19, Lippincott’s

2. OVERALL NITROGEN METABOLISM
• Nitrogen enters the body in a variety of compounds esp. dietary protein.
• Nitrogen leaves the body as urea, ammonia, and other products derived
from amino acid metabolism.
• AMINO ACID POOL
• All of the FAAs present in cells and ECF throughout the body
• Supplied by 1) degrad. Of body proteins; 2) degrad. Of dietary proteins; 3)
synthesis of non-essential (ne) aa from intermediates of metabolism
• Depleted by 1) synthesis of body proteins (bp); 2) synthesis of N-containing
small molecules purines/pyrimidines/creatinine etc.; 3) conversion of aa
into glucose/glycogen/ketone bodies/CO2+H2O

3. • PROTEIN TURNOVER
• The phenomenon of simultaneous synthesis and degradation of
proteins.
• For most proteins, conc. Is determined mainly by reg. of their
synthesis and degradation being not so reg.
• rate= 300-400g protein being degraded and resynthesized each day

4. PROTEIN DEGARADTION
• a. Ubiquitin–proteasome proteolytic pathway:
• Selective; atp dependent;
• Selection of protein to be degraded; covalent attachment of protein
with UBIQUITIN (4-molecules)>> catalyzed by 3 enzymes using ATP>>
producing a polyubiquitinated protein; recognized by PROTEASOME
complex>> unfolds, deubiquitinates, and cuts the target protein into
fragments>> fragments then further degraded by cytosolic proteases
to aas

6. PROTEIN DEGRADATION
• ATP-independent degadative enzyme system of the lysosomes
• Non-selective; atp-independent
• Lysosomes use acid hydrolases to no selectively degrade
intracellular proteins (“autophagy”) and extracellular proteins
(“heterophagy”), such as plasma proteins, that are taken into the cell
by endocytosis

7. DIGESTION OF DIETARY PROTEINS 247
• A. Digestion by gastric secretion
• Digestion begins in stomach by gastric juice containing HCl (parietal
cells) and pepsinogen (chief cells)
• HCl>> minor role in protein digestion due to dilution but denatures
protein and kills microbes
• Pepsinogen>pepsin (by HCl or pepsin itself)> hydrolyses proteins to
polypeptides and some faa
• Pepsin>> endopeptidase

8. • B. Digestion by pancreatic enzymes
• Include both endopeptidases and exopeptidases; trypsin; chymotrypsin;
elastase; carboxypeptidase; produce oligopepides and faas
• All are zymogens; secretion regulated by cholecystokinin and secretin
• All these enzymes have different specificity for the amino acid R-groups
adjacent to the susceptible peptide bond see figure
• Enter peptidase activates trypsin and trypsin then activates all other
pancreatic zymogens
• Bicarbonate raises pH

9. • C. Diges tion of oligopeptides by enzymes of the s mall intes tine
• Aminopeptidase(exopeptidase); di,tri-peptidases
• Converts oligopeptides to further small peptides and faas
•

10. • D. Abs orption of amino acids and s mall peptides
• Faas> enterocytes via sodium linked co trapsport
• Di, tri peptides> enterocytes via proton linked transport; in cells they are
then converted into faas
• Faas enter portal system (hepatic portal vein etc.) via facilitated diffusion
• Faas> liver > degradation or enter circulation
• From circulation faas enter body cells via active transport systems (7-
different systems are known for their transport)
• Cystinuria

11. REMOVAL OF NITROGEN FROM AMINO ACIDS
• In form of a-amino acids; aa can’t be degraded; remove NH2- group
then aa can be metabolized
• This removal involves
• Trans amination: the funneling of amino groups to glutamate
• Oxidative deamination of amino acids
• These processes provide aspartate and ammonia for synthesis of urea
in urea cycle

12. • A. Trans amination: the funneling of amino groups to glutamate
• Transfer of amino group (ag) from aa to akg (alpha-ketoglutarate);
• aa>> aka(alpha-keto acid) and akg>> glutamate (glm)
• Glm produced may either be oxidatively deaminized or used in the synthesis of
neaa
• Reactions catalyzed by aminotransferases; ALT & AST; all reversible reactions
• ALANINE AMINO TRASNFERASE; transfers NH2- from alanine to akg
• Alanine>> pyruvate & akg>> glm
• ASPARTATE AMINOTRANSFERASE; Transfers NH2- from glm to OAA
• Glm>> akg & oaa>> aspartate
• ALT AND AST USED AS DIAGNOSIC BIOMARKER FOR LIVER DISEASES

14. • B. Oxidative deamination of amino acids
• the simultaneous loss of ammonia coupled with the oxidation of the carbon
skeleton
• Removal of NH2- group (primarily from glutamate) and formation of NH3 and aka
• NH3 liberated exists as NH4+
• Glutamate >> oxidative deamination>> NH3 + akg
• Reaction catalyzed by GDH and coenzyme NADH + H+
• Akg >> reductive amination >> glutamate
• Reaction catalyzed by GDH and coenzyme NADPH + H+
• DIRECTIONS OF RXs
• Meal> high protein> high glutamate> aa degradation> NH3 synthesis
• High NH3 levels> glutamate (aa) synthesis
• ADP (low energy levels) > aa degradation> NH3 synthesis by GDH
• GTP (high energy levels) > glutamate synthesis by GDH

16. • C. Transport of ammonia to the liver
• Two mechanisms; both involving muscles a) glutamine formation b) alanine
formation
• A) NH3 + Glutamate> GLUTAMINE (in muscle) > glutamine transported to LIVER>
where it is cleaved by Glutaminase into glutamate and NH3. NH3 is used in
urea synthesis.
• B) glucose> pyruvate via glycolysis in muscle; succinyl coA to pyruvate also in
muscles; pyruvate is then converted into ALANINE by transamination catalyzed by
ALT; alanine transported out from muscles through blood into LIVER.
• In liver alanine is converted to pyruvate via transamination by ALT producing
glutamate.
• Glutamate produced in both mechanisms is acted upon by GDH and is
converted into akg and NH3. again NH3 is used for UREA synthesis

19. UREA CYCLE
• One nitrogen from free ammonia (by oxidative deamination of glm) and other
from aspartate
• Carbons of urea are derived from HCO3-
• 1st 2 reactions occur in mitochondrial matrix while the remaining occur in cytosol
• 1. Formation of carbamoyl phosphate :
• Carbamoyl phosphate synthase I (CPS-I) converts one molecule of NH3 and HCO3-
using 2 molecules of ATP to form 1 molecule of carbamoyl phosphate (CP)
• CPS-I requires N-acetylglutamate as an allosteric activator
• 2. Fo rmatio n o f c itrulline :
• Ornithine transcarboxylase (OTC) transfers carbamoyl portion of CP to ornithine
to form citrulline.
• Inorganic phosphate is released
• Ornithine and citrulline move across mitochondrial membranes via co
transporters

20. • 3. Synthesis of argininosuccinate :
• Argininosuccinate synthetase combines citrulline with aspartate (containing 2nd
nitrogen of urea) to form argininosuccinate
• 1 ATP is used and converted into AMP; last ATP of urea cycle; total 3 ATPs are utilized
• 4. Cleavage of argininosuccinate:
• Argininosuccinate lyase cleaves argininosuccinate into fumarate and arginine.
• Arginine serves as a precursor of urea
• Fumarate is hydrated into malate; malate enters mitochondria via malate-aspartate shuttle
and is oxidized into OAA by ongoing TCA-cycle in matrix.
• OAA may be used
• in gluconeogenesis to form glucose
• For formation of aspartate by transamination carried out by AST

21. • 5. Cleavage of arginine to ornithine and urea:
• Arginase hydrolyses arginine to ornithine and urea
• Arginase is present only in liver
• 6. Fate of urea:
• Urea diffuses from liver into blood and is transported to the kidneys where it is excreted
• Part of urea is left unexcreted and goes to intestine via blood where bacterial UREASE
cleaves it into NH3. this ammonia is removed in feces.
• Kidney failure of urea excretion> more urea in blood> more urea to reach intestine> more
bacterial urease activity> more NH3 production> hyperammonemia> may be treated with
antibiotics to kill intestinal bacteria containing UREASE
• Overall stoichiometry of the urea cycle
• Aspartate + NH3 + HCO3– + 3 ATP + H2O →
urea + fumarate + 2 ADP + AMP + 2 P i + PP i

23. METABOLISM OF AMMONIA
• A. Sources of ammonia
• 1. From glutamine: produced from catabolism of branched chain aa; transported via blood to
liver kidneys and intestine
• Liver and kidneys produce NH3 from glutamine by the action of glutaminase and GDH.
• NH3 produced is converted into urea in case of liver and excreted
• in case of kidneys, NH3 is excreted as NH4+ in urine therefore plays role in maintaining acid-base balance
• In intestine intestinal mucosal cells produce NH3 by the action of intestinal glutaminase on
glutamine. This ammonia either enters blood or is removed via feces
• 2. From bacterial action in the intestine: urea produced in liver enters blood and reaches
kidneys and to some extent intestine. In intestine, bacterial urease converts urea to NH3. This
ammonia either enters blood or is removed via feces
• 3. From amines :
• 4. From purine s and pyrimidines :

24. • B. Transport of ammonia in the circulation
• Ammonia in its free form can be vey toxic for CNS. Therefore it is
transported in blood in form of glutamine or alanine rather than as free
NH3
• NH3 can be converted into urea for safe disposal via kidneys
• Or NH3 can be converted into glutamine by combining glutamate and NH3
in the presence of glutamine synthase. Glutamine is a safe transport and
storage form of NH3
• Glutamine formation occurs mostly in muscles but is of significant
importance in CNS to avoid toxic levels of NH3

25. HYPERAMMONEMIA
• Normal NH3 levels 5-35 micromole/liter
• Elevated levels due either to liver disease (acquired
hyperammonemia) or genetic defects (congenital hyperammonemia)
can raise the NH3 blood levels above 1000 micromoles/liter
• Elevated concentrations of ammonia in the blood cause the
symptoms of ammonia intoxication, which include tremors, slurring of
speech, somnolence (drowsiness), vomiting, cerebral edema, and
blurring of vision.
• At high concentrations, ammonia can cause coma and death