1. Special aspects of renal metabolism Mahmoud A. Alfaqih BDS PhD Jordan University of Science and Technology

2. Outline  Transport of amino acids into cells  Metabolism of urea  Metabolism of creatinine  Clinical significance of urea and creatinine measurements in the plasma

3. Overview  Unlike fats and carbohydrates, amino acids are not stored by the body  Amino acids must be obtained from the diet, synthesized de novo, or produced from normal protein degradation  Any amino acids in excess of the biosynthetic needs are rapidly degraded  Catabolism of amino acids involves two phases: 1. The removal of the α-amino groups (by transamination and subsequent oxidative deamination) forming ammonia and the corresponding α-keto acid 2.The carbon skeletons of the α-ketoacids are converted to intermediates of energy producing pathways  A portion of ammonia is excreted unchanged in urine, the remaining is converted into urea and excreted in urine

4. TRANSPORT OF AMINO ACIDS INTO CELLS Special aspects of renal metabolism

5. Transport of amino acids into cells  The extracellular concentration of free amino is significantly lower than inside the cells  The concentration gradient is maintained through active transport (requires ATP) that moves amino acids into cells  Seven different transport systems are known that have overlapping specificities for different amino acids  The small intestine and the proximal tubule of the kidney share the same transport systems for amino acid uptake  A defect in any of the systems results in an inability to absorb amino acids into the gut and into the kidney tubules

6. Clinical correlation: Cystinuria  An inherited disorder caused by a defect in the uptake system of dibasic amino acids (Cystine, Ornithine, Arginine, Lysine)  All four amino acids appear in the urine.  Cystinuria occurs at a frequency of 1 in 7,000 individuals.  The most common genetic error of amino acid transport.  The disease is characterized by the precipitation of cystine to form kidney stones which can block the urinary tract.  Hydration is an important part of treatment.

8. METABOLISM OF UREA Special aspect of renal metabolism

9. General structure of amino acids

11. Removal of nitrogen from amino acids: Overview  The presence of the α-amino group keeps amino acids safely locked away from oxidative breakdown  Removing α-amino group is obligatory in the catabolism of all amino acids  Once removed, nitrogen can be incorporated into other compounds or excreted, with the carbon skeletons metabolized  Transamination and oxidative deamination will provide ammonia and aspartate (sources of urea nitrogen)

12. Transamination: the funneling of amino groups into Glutemate  The first step in the catabolism of most amino acids is the transfer of their α- amino group to α-ketoglutarate  The products are an α-keto acid (derived from the original amino acid) and glutamate  Glutamate produced by transamination can be: 1.Oxidatively deaminated (Glutemate dehydrogenase) 2.Used as an amino group donor in the synthesis of amino acids  Transamination is catalyzed by a group of enzymes known as aminotransferases  Aminotransferases are found in the cytosol and mitochondria of cells throughout the body

13. Substrate specificity of aminotransferases  Each aminotransferase is specific for one or a few amino group donors  Aminotransferases are named after the specific amino group donor  Acceptor is always α-ketoglutarate  Two clinically important aminotransferase reactions are catalyzed by alanine aminotransferase (ALT) and aspartate aminotransferase (AST)

14. Alanine aminotransferase (ALT)  Formerly called glutamate-pyruvate transaminase (GPT)  The enzyme catalyzes the transfer of the amino group of alanine to α-ketoglutarate, resulting in the formation of pyruvate and glutamate  Bi-directional reaction  During amino acid catabolism, functions in the direction of glutemate synthesis  Glutemate is a collector of amino groups from alanine

15. Alanine aminotransferase

16. Aspartate aminotransferase (AST)  Formerly called glutamate-oxaloacetate transaminase (GOT)  AST is an exception to the rule that aminotransferases funnel amino groups to form glutamate  AST transfers amino groups from glutamate to oxaloacetate, forming aspartate (during catabolism)  Aspartate is used as a source of nitrogen in the urea cycle

17. Aspartate aminotransferase

19. Diagnostic value of ALT and AST  Aminotransferases are intracellular enzymes, with the low levels found in the plasma  Elevated plasma levels of aminotransferases indicate damage to cells rich in these enzymes  Physical trauma or a disease process cause cell lysis, causing release of intracellular enzymes into the plasma  AST and ALT are of particular diagnostic value when found in the plasma

20. Liver disease  Plasma AST and ALT are elevated in nearly all liver diseases  ALT and AST are particularly high in severe viral hepatitis, toxic injury, and prolonged circulatory collapse  ALT is more specific than AST for liver disease  AST is more sensitive because the liver contains larger amounts of AST

21. Non-hepatic disease  ALT and AST may also be elevated in non-hepatic disease, such as myocardial infarction and muscle disorders

22. Glutamate dehydrogenase: the oxidative deamination of amino acids  Oxidative deamination by glutamate dehydrogenase results in the release of ammonia  This reaction occurs in the liver and kidney  The reaction produces α-ketoglutarate and ammonia (source of nitrogen in urea synthesis)

24. Glutamate dehydrogenase Direction of reaction  It depends on the relative concentrations of glutamate, α-ketoglutarate, and ammonia, and the ratio of oxidized to reduced coenzymes  The reaction can also be used to synthesize glutemate from α-ketoglutarate Allosteric regulators  ATP and GTP are allosteric inhibitors  ADP and GDP are allosteric activators

25. Transport of ammonia to the liver  Two mechanisms of transport of ammonia from the peripheral tissues to the liver 1.In the form of glutamine  Used by most tissues.  Uses glutamine synthetase to combine ammonia with glutemate to form glutamine  Glutamine transported to the liver  Glutamine is cleaved in the liver into glutemate and ammonia by glutaminase

26. Transport of ammonia to the liver 2. In the form of alanine  Used primarily by the muscle.  Transamination of pyruvate into alanine.  Alanine is transported to the liver by the blood.  In the liver, alanine is converted back into pyruvate releasing free ammonia (transamination)  Pyruvate can be used by the liver to make glucose  The above pathway is called glucose-alanine cycle

28. Urea cycle  Urea is the major disposal form of amino groups derived from amino acids.  Urea accounts for 90% of the nitrogen-containing components of urine.  One nitrogen of the urea molecule is supplied by free NH 3, and the other nitrogen by aspartate  Glutamate is the immediate precursor of both ammonia and aspartate

29. Urea Cycle

31. Fate of urea  Urea diffuses from the liver, and is transported in the blood to the kidneys, where it is filtered and excreted in the urine.  A portion of the urea diffuses from the blood into the intestine, and is cleaved to CO 2 and NH 3 by bacterial urease  This ammonia is partly lost in the feces, and is partly reabsorbed into the blood.

32. Overall stoichiometry of the urea cycle  The synthesis of urea is irreversible, with a large, negative ΔG.  One nitrogen of the urea molecule is supplied by free NH 3, and the other nitrogen by aspartate.  Glutamate is the immediate precursor of both nitrogens: Through oxidative deamination of glutemate dehydrogenase (releases free ammonia) Transamination reaction with oxaloacetate which produces Aspartate

34. Regulation of urea cycle  Formation of carbamoyl phosphate is the rate limiting step in urea cycle, catalyzed by carbamoyl phosphate synthetase I  N-Acetylglutamate is an essential activator for carbamoyl phosphate synthetase I  N-Acetylglutamate is synthesized from acetyl coenzyme A and glutamate by N-acetylglutamate synthase  Arginine is an activator of N-acetylglutamate synthase

36. Hyperammonemia  Levels of serum ammonia are normally low (5–50 µmol/L)  The capacity of the hepatic urea cycle exceeds the normal rates of ammonia generation  Genetic defects of the urea cycle, or liver disease will cause blood levels of ammonia to rise  Hyperammonemia is a medical emergency, because ammonia has a direct neurotoxic effect on the CNS

37. Acquired hyperammonemia  Liver disease is a common cause of hyperammonemia in adults  Common causes include: viral hepatitis, ischemia, hepatotoxins, cirrhosis, biliary obstruction leading to collateral circulation around the liver

38. Hereditary hyperammonemia  Genetic deficiencies of each of the five enzymes of the urea cycle have been described  Ornithine transcarbamoylase deficiency is the most common (X- linked)  Failure to synthesize urea leads to hyperammonemia during the first weeks following birth  Treatment includes limiting protein and administering compounds that bind covalently to amino acids and cause their excretion

40. CREATININE METABOLISM Special aspect of renal metabolism

41. Creatine: Overview  Creatine phosphate (phosphocreatine) is found in muscle.  Creatine Phosphate is a high-energy compound that can donate a phosphate group to ADP to form ATP.  Creatine phosphate provides a small, rapidly mobilized reserve of high-energy phosphates.  Creatine phosphate is used to maintain the levels of ATP during the first few minutes of intense contraction.  The amount of creatine phosphate is proportional to the muscle mass

42. Synthesis of Creatine  Creatine is synthesized from glycine and the guanidino group of arginine, plus a methyl group from SAM  Creatine is reversibly phosphorylated to creatine phosphate by creatine kinase, using ATP  N.B: The presence of creatine kinase in the plasma is used in the diagnosis of myocardial infarction

44. Degradation of Creatine  Creatine and creatine phosphate spontaneously cyclize to form creatinine (Excreted in urine) The amount of creatinine excreted can be used to estimate muscle mass When muscle mass decreases (paralysis, muscular dystrophy), the creatinine content of the urine falls. Any rise in blood creatinine is an indicator of kidney malfunction, because creatinine is rapidly removed from the blood and excreted.