1. # 3 UREA CYCLE Reactions of the Urea Cycle Enzyme Regulation of the Urea Cycle Nutritional Regulation of Urea Synthesis Urea Cycle Disorders & Treatment

2. Urea Cycle 1.GDH is the major agency responsible for ammonium production. 2.Ammonium is toxic (N = 15 - 40  M, max 70  M) Urine: organic acids and orotic acid 3. Liver: Principal site but also in small intestine 4.Excretion NH 4 + by kidneys important for acid-base balance but Normally 80-90% N  urine as urea. 5.Hyperammonium >500  M plasma [NH 4 + ] = TOXIC related to inborn errors of metabolism (genetic defects) as well as induced (liver failure) Usually detected in the newborn period. Blood: measure ammonium, AA, lactate

3. Nitrogen-containing components of normal urine

4. Urea Cycle 1.The urea cycle was the first metabolic process to be described as a cycle by Sir Hans Krebs who also described the TCA cycle. 2.Role of Urea cycle: rid the body of toxic NH 4 + therefore permitting the use of AA as an energy source. 3.Liver major site of urea synthesis, major source of arginase, (small amounts in small intestine) and is the only tissue with the complete set of all 5 enzymes required 4.Other tissues have enzymes for reactions (iii) and (iv) only to make ARG or NO (important in blood pressure, neuro transmission, macrophage antibacterial action)

5. Urea Cycle

6. I.Compartmentation: mitochondria (rxn 1&2) cytosol (rxn 3-5) II. CP = 20% mitochondrial protein III. Cyclic inter conversion of ornithine / arginine. IV. Ornithine is used in the same way as is oxaloacetate in the TCA cycle. It is the carrier of a substituent group that undergoes modification and is subsequently split off.

7. 1. 2 N per urea molecule: 1 NH4+ (start) + 1 transferred from ARG 2. 4 high energy phosphate:2 ATP  ADP + Pi 1 ATP  AMP + Ppi Therefore 2 ATP / amino (N) group Overall catabolism: Catabolize 1 Leu  32 ATP (from TCA cycle) Make urea from N  2 ATP NET ENERGY 30 ATP produced Mathematics of equation

8. Short term Regulation: CPS1 1.NAG(N-acetyl glutamate), a positive allsoteric regulator is absolutely required. Alters enzyme conformation 2.NAG is synthesized in liver mitochondria from acetyl CoA and GLU FA or pyruvate  acetyl CoA Diet or tissue proteins  AA  GLU and ARG Acetyl CoA + GLU  NAG (enzyme = NAG synthase) 3.NAG synthesis is markedly stimulated by ARG (allosteric) but not completely dependent (  V max) therefore  AA   NAG 4.Hyperammonemia that develops with acidemia  NAG synthesis inhibition (propionic acidemia, isovaleric acidemia, nethylmalonic acidemia) due to competition for CoA (see figure)

9. Regulation via NAG

10. Regulation through Mg 2+ (i) Mg 2+ : CPS1 dependent Mg 2+ ( both ATP and free) Therefore changes in mitochondrial citrate can affect reaction since citrate chelates Mg 2+ (ii) Zn 2+ is present in mitochondria Zn 2+ decreases CPSI activity in vitro However, AA (ornithine) can chelate Zn therefore preventing inhibition of CPS1. (iii) CPS1  20% total liver protein (0.4 mM)  [substrate] eg NH 4 +, HCO 3, ATP - Mg 2+, NAG Therefore not operating at maximum capacity and important to inhibit to keep some NH 4 + available to make GLN

11. Nutritional Regulation “long term regulation” (i) Five Urea Cycle enzymes & NAG synthase all  with low P diets &  with high P diets Therefore regulated nutritionally (over the long term) (ii) Note also  during starvation due to  AA catabolism therefore although muscle and liver protein  the level of these enzymes  due to increased urea synthesis -increased enzyme synthesis -decrease enzyme degradation (iii) Changes take place over 3-7 days.

12. Urea Cycle Disorders Prevalence of disorders: 1/30,000 live births but may be more since some die undiagnosed. Mode of inheritance = usually autosomal recessive (2 - ve genes) OTC (most common) X-linked, heterozygotes generally asymptomatic (i) Deficiency enzymes rxn 1-4  hyperammonemia. In general: concentration of AA metabolites  proximal &  distal. (ii) In all disorders:  NH4,  GLN,  ALA (iii) Less severe defects: (partial deficiencies)  less side effects, manifested only in later childhood or adulthood.

13. Defects of Urea Cycle ↑ orotic acid III IV V

14. Presentation Severe Illness : First week Usually normal first 24h Symptoms of hyperammonemia within 1-3 days Include: Feeding intolerance Vomiting Lethargy Irritability Respiratory Distress (hyperventilation) Seizures Coma

15. Outcome Mortality Improvements in treatment have increased 1 year survival rate. Once past the neonatal period, long term survival rate = 50% OTC (Type II) 75% CPS (Type I) 95% AS and AL (Steps 3+4) Morbidity 75% mental retardation (mean IQ 50), Seizure disorders, Visual deficits (proportional to extent of  NH 4 ), Protein intolerance Brain:  NH 4 causes increased permeability and TRP    serotonin  behavior abnormalities  quinolininc acid  neuronal injury Also with type V block   Arg but ~ NH 4 +  severely retarded

16. Treatment: Reduce N Intake Provide sufficient for growth (need EAA ) but avoid  NH 4 using a high calorie low P diet Provide ARG supplement (except type V) since ARG synthesis  therefore  growth,  N incorporation into AA therefore  NH 4 ARG also  NAG synthase therefore  CPSI (if not type I) ARG also  ornithine (ARG is precursor) especially important in type III and IV (where citrulline & arginosuccinate are lost in urine) ARG also  alternate NH 4 + excretion (through alternate pathway) Replacement with EAA (as  keto acids to limit N intake) which can be formed into AA through transamination

17. Treatment (cont’d) 1. Compounds to Conjugate AA:(  urea load) (see Diagram) Benzoate: combines with GLY to generate hippurate  urine Phenylacetate: +GLN to produce phenylacetyl GLN  urine 2. NAG Permeable Analog: N carbamoyl glutamate enters mitochondrial. 3. Hemodialysis used to remove both AA & NH 4 during hyperammonemia coma

18. Treatment: Stimulate Alternate Pathways Stimulate Alternate Pathway: ARG  ornithine  citrulline  arginino succinate Citrulline & argino succinate can be secreted in urine

19. Future (i) Enzyme Replacement Therapy (Liver Transplant) but expensive and lack donors (ii) Gene Therapy  In mice to date, In OTC deficient mouse transfection using adeno virus vector is successful (iii) Diagnosis Molecular Diagnostics (RFLP) can reveal genetic defects by prenatal diagnosis when indicated. Direct enzyme determination in amniocytes or chorionic vilus biopsy to determine presence/absence enzyme Reactive or anticipatory treatment if defect suspected

20. Case #3 Discussion A 6-month-old infant began to vomit occasionally and ceased to gain weight. At age 8½ months he was readmitted to the hospital. Routine examination and laboratory tests were normal, but after 1 week he became habitually drowsy, his temperature rose to 39.4oC, his pulse was elevated, and his liver was enlarged. The electroencephalogram was grossly abnormal. Since the infant could not retain milk given by gavage feeding, intravenous glucose was administered. He improved rapidly and came out of the coma in 24 hours. Analysis of his urine showed abnormally high amounts of glutamine, uracil & orotic acid but ↓ urea, which suggested a high blood ammonium concentration. This was confirmed by the laboratory.

21. Discussion: 1. Hereditary hyperammonemia can result from defects in genes for urea cycle enzymes. Which enzymes might be affected? 2. Considering the data (↑ uracil & orotic acid) which enzyme may be defective in this patient? 3. Why was the urine glutamine concentration elevated? 1. Hyperammonemia is characteristic of all steps (including NAG synthase) Most frequent OTC 2.  N BUN ( blood urea N), ALSO  uracil (&  orotic acid) due to  carbamoyl phosphate which leaks from mito  cyto  increased pyrimidine synthesis. Unusual: clinical symptoms slow (6 months old) 3 Why? Exceeds kidneys ability GLN  GLU + NH 4 +

22. Cont’d 4. Offer a genetic explanation for the observation that this disease is usually lethal in males but not in affected females. 5. This patient was treated using procedures available at the time. He was given a daily diet of 1.5 g of protein/kg body weight. After 2 years on this diet, his height and weight were judged to be normal for his age. What is the effect of diet on a growing child in terms of nitrogen balance? 6. How would you treat a similar patient today? 4. Disease is x linked, men have only 1 X chromosome, women have two X chromosomes. Therefore more severe in men than women (usually). 5. Growing child requires increased N, therefore load on urea  P diet. Balance between P restriction (prevent NH 4 + ) and enough for growth. Not usually sufficient for patients -ve OTC 6. Hemodialysis / transfusion asap (prevent brain damage) IV benzoate, phenylacetate to act as NH 4 traps