Detoxification of endogenous and exogenous compounds

1. Detoxification ofendogenous and exogenouscompounds mirka.rovenska@lfmotol.cuni.cz

2. A) Detoxification of ammonia • Ammonia originates in the catabolism of amino acids that are primarily produced by the degradation of proteins – dietary as well as existing within the cell: • digestive enzymes • proteins released by digestion of cells sloughed-off the walls of the GIT • muscle proteins • hemoglobin • intracellular proteins (damaged, unnecessary)

3. transamination oxidative deamination urea cycle Nitrogen removal from amino acids

4. Ammonia has to be eliminated: • Ammonia is toxic, especially for the CNS, because it reacts with-ketoglutarate, thus making it limiting for the TCA cycle  decrease in the ATP level • Liver damage or metabolic disorders associated with elevated ammonia can lead to tremor, slurred speech, blurred vision, coma, and death • Normal conc. of ammonia in blood: 30-60 µM

5. Transamination • Transfer of the amino group of an amino acid to an -keto acid  the original AA is converted to the corresponding -keto acid and vice versa:

6. L-alanine L-aspartate -ketoglutarate glutamate oxalacetate pyruvate

7. Transamination is catalyzed by transaminases (aminotransfera-ses) that require participation of pyridoxalphosphate: amino acid pyridoxalphosphate Schiff base

8. Principal transaminations: • Alanine transaminase (in the muscle): AA + pyruvate  -keto acid + Ala • Glutamate transaminase: AA + -ketoglutarate -keto acid + Glu • Aspartate transaminase: AA + oxaloacetate  -keto acid + Asp • Transaminations are usually reversible  the actual direction depends on the concentrations of reactants

9. Result: • Most of transaminases use -ketoglutarate as an -keto acid, to a lesser extent oxalacetate, thus producing mainly Glu and Asp • Glu is either oxidatively deaminatedreleasing ammonia that – in the liver – enters the urea cycle, or used for syntheses • Aspartate enters the urea cycle

10. Oxidative deamination of Glu • In mitochondria • Glu + NAD(P)+ + H2O → NAD(P)H + H+ + NH4+ + -ketoglutarate • Catalyzed by glutamate dehydrogenase that is capable of using both NAD+ and NADP+ • Reaction is reversible – either produces Glu, or releases ammonia, depending on the concentrations of reactants • Ammonia enters the urea cycle where it is converted to urea

11. Transport of nitrogen – 1) as Gln • In the tissues, ammonia is built into Gln by glutamine synthetase: Glu + ATP + NH4+  Gln + H2O + ADP + P • Gln is transported to the liverand kidneyand deaminated byL-glutaminase: • Glu can be oxidatively deaminated, ammonia is excreted by the kidneys or converted to urea in the liver Amide nitrogen, not the -amino nitrogen is removed!

13. Gln in the kidney: • A portion of Gln can be taken up by the kidney; another portion of Gln is produced by the kidney itself • Ammonia released by the glutaminase reaction in the kidney diffuses into the urine instead of entering the urea cycle • These processes participate in the regulation of the acid-base balance and ofpH of the urine

14. Liver Muscle Transport of nitrogen – 2) as Ala • Mainly by the muscle • i.a. in the glucose-alanine cycle:

15. In the fed state, AA released by digestion travel through the hepatic portal vein to the liver and other tissues, where they are used primarily for the synthesis of proteins (in the liver, particularly for the synthesis of plasma proteins):

16. Gln and Ala are the major carriers of nitrogen • Upon fasting, some tissues (brain, skeletal muscle, kidney) oxidize Val, Leu, Ile and incorporate nitrogen into Gln, Ala • Gln, Ala and other AA carry nitrogen to the liver, kidney, gut, and cells with rapid turnover rate (leukocytes) for biosyntheses (Nt), oxidation, or synthesis of glucose and ketone bodies • The unused nitrogen is carried as Ala to the liver to the urea cycle

17. Detoxification of ammonia • a) ammonia is built into Glu by glutamate dehydrogenase andGln by glutamine synthetase: -ketoglutarate + NH4+ + NAD(P)H+H+  Glu + H2O + NAD(P)+ Glu + ATP + NH4+  Gln + H2O + ADP + P • Glu, Gln can then be used for syntheses: • Glu – for the synthesis of Gln, Pro, Ala, Asp • Gln – for the synthesis of purines and pyrimidines • Transamination Glu + oxalacetate → Asp + 2-oxoglutarate supplies the urea cycle with Asp!!! • b) the urea cycle converts ammonia to urea…PRINCIPAL

18. serine dehydratase – NH3 Sources of ammonia for the urea cycle: • Oxidative deamination of Glu, accumulated in the liver by the action of transaminases and glutaminase • Glutaminase reaction releases NH3 that enters the urea cycle in the liver (in the kidney, it is excreted into the urine) • Catabolism of Ser, Thr, and His also releases ammonia: • Bacteria in the gut also produce ammonia By analogy: Thr to α-ketobutyrate

19. Urea cycle • In the liverin 2 compartments: mitochondrial matrix + cytoplasm • In the mitochondrial matrix, oxidative deamination of Glu releases ammonia that is converted to carbamoyl phosphate: NH4+ +HCO3- + 2 ATP  2 ADP +P+ • In mitochondria, carbamoyl phosphate reacts with ornithine, yielding citrulline, which is transported to the cytoplasm • Ornithine is regenerated by step 5 and transported back to mitochondria carbamoyl phosphate

20. Fumarate ← Glu + oxalacetate

21. 3 moles of ATP are required for the formation of 1 mole of urea: • 2 for the formation of carbamoyl phosphate • 1 for the formation of argininosuccinate

22. Regulation by N-Ac-Glu • Carbamoyl phosphate synthetase I (CPSI) is activated by N-acetylglutamate: • N-Ac-Glu is synthesized from Glu and AcCoA which can be stimulated by Arg • When AA breakdown rises, conc. of Glu and Arg increase the concentration of N-Ac-Glu is also increased  activation of CPS I  stimulation of the urea cycle

23. Deficiencies of the urea cycle enzymes • Lead to elevated Gln and ammonia levels in the circulation • 1) N-acetylglutamate synthetase • administration of carbamoyl glutamate (also activates CPSI) • 2) CPSI: • administration of benzoate and phenylacetate→hippurate and Phe-Ac-Gln are excreted in the urine:

24. 3) Ornithine transcarbamoylase – the most common deficiency • the same treatment as in the case 2) • 4) Argininosuccinate synthetase accumulation of citrulline in the blood and excretion in the urine (citrullinemia) • supplementation with Arg necessary • 5) Argininosuccinate lyase: • treatment as in the case 2) + supplementation with Arg • 6) Arginase (rare)  Arg accumulates and is excreted • administration of benzoate + low protein diet including essential AA (but excluding Arg) or their keto analogs • In all cases, the low nitrogen diet is applied

25. Other nitrogenous degradation products excreted in the urine • Creatinine – produced from creatine phosphate: • Uric acid – degradation product of the purine bases

26. B) Metabolism of xenobiotics • Drugs, preservatives, pigments, pesticides … • Predominantly in the liver, also in the intestines, kidney, lungs • Involves two phases

27. Phase 1 • Incorporation of new groups or alteration of groups that are already present in the molecule • In the endoplasmic reticulum (ER) • Result: • increase in the polarity (supports excretion) • change in biological activity: • A) decrease in the biological activity (toxicity) • B) activation: some compounds only become biologically active once they have been subjected to phase 1

28. Potential toxic effects ofactivated compounds • Cytotoxicity – e.g. by covalent binding to proteins • Binding to a protein, thus altering its antigenicity antibodies are produced that can damage the cell • Carcinogenesis – phase 1can convert procarcinogens (e.g. benzo[]pyren) to carcinogens. • Epoxid hydrolase (in ER) can convert reactive, mutagenic and/or carcinogenic epoxides to less reactive diols: epoxide diol

29. Reactions of phase 1: • Hydroxylation • Epoxide formation • Reduction of carbonyl-, azo-, or nitro- compounds • Dehalogenation

30. Hydroxylation • Chief reaction of the phase 1 • Catalyzed by cytochrome P450s: • in humans: ~60 isoenzymes; the most abundant: CYP3A4 • monooxygenases: RH + O2 + NADPH + H+ ROH + H2O + NADP+ • Electrons from NADPH+H+ are transferred to NADPH-cytochrome P450 reductase, then to cytochrome P450 andto oxygen→one oxygen atom is inserted into the substrate • They metabolize not only xenobiotics but also endogenous compounds, e.g. some steroids, eicosanoids

31. CYP3A4 4…isoform number within the subfamily CYP = cytochrom P4503…family A…subfamily Isoforms of cytochrome P450 • Hemoproteinsin the endopl. reticulum,inner mitoch. membrane • Most abundant in the liver and small intestine followed by lungs • Nomenclature based on the AA sequence identity: • Some exist in polymorphic forms, some of which exhibit low activity  accumulation of the corresponding xenobiotic • Some are involved in metabolism of polycyclic aromatic hydro-carbons (PAHs), thus playing a role in carcinogenesis

32. Most isoforms are inducible: • E.g. by phenobarbital and other drugs, but also by their own substrates • Mechanism: mostly increased transcription • Can lead todrug interaction: • induction of the particular isoform by the drug 1 (e.g. phenobarbital) can speed up metabolism of the drug 2 (e.g. warfarin) by this isoform  it is necessary to increase the dose of the drug 2

33. Metabolism of ethanol • The other route (~10-20%): by the cyt P450 isoform CYP2E1: CH3CH2OH + NADPH+H+ + O2 → NADP+ + 2 H2O + CH3CHO Acetaldehyde can enter the blood and damage tissues. • CYP2E1 is induced by ethanol and metabolizes alsosome carcinogenic components of tobacco smoke! – mainly in the liver Most of acetate enters the blood and, mainly in the ske-letal muscle, is activated to acetyl-CoA → TCA cycle

34. Phase 2 – conjugation • Products of phase 1 are conjugated with: • glucuronate • sulphate • glutathione • Conjugation renders them even more water-soluble and eventually even less active; conjugates are excreted with the bile (conjugates with Mr 300) or urine (Mr  300)

35. Glucuronidation • UDP-glucuronic acid is the glucuronate donor: • Glucuronate can be attached to oxygen (O-glucuronides) or nitrogen (N-glucuronides) groups • Excreted as glucuronides are: benzoic acid, meprobamate, phenol, and also endogenous compounds – bilirubin, steroids glucuronate

36. Bilirubin excretion • Bilirubin is the product of heme catabolism: heme

37. M: methyl, V: vinyl, CE: carboxyethyl (propionic) transported to the liver bound to albumin

38. heme→ biliverdin → bilirubin transport to the liver (albumin) conjugation with glucuronate  bilirubin diglucuronide secreted into the bile bacteria in the small intestine release bilirubin from diglucuronide and convert it to colourless urobilinogens a small fraction is reabsorbed and re-excreted through theliver into the bile a small fraction is excreted into the urine by the kidney most of them are oxidi-zed to pigments and excreted in the faeces (urobilin, stercobilin)

39. Sulfation • Some alcohols, arylamines, phenols, but also glycolipids, steroids • Sulfate donor: PAPS (3´-phosphoadenosine-5´-phosphosulfate):

40. Conjugation with glutathione • Glutathione (GSH) = -glutamylcysteinylglycine: • Conjugation with GSH: G–S–H + R  G–S–R + H+(R…electrophilic xenobiotic) • Conjugation with GSH prevents binding of distinct xenobiotics to DNA, RNA, or proteins, and subsequent cell damage!

41. Metabolism of glutathione conjugates: • Glutamyl and glycinyl are removed from GSH • an acetyl group (donated by acetyl-CoA) is added to the amino group of the Cys moiety • mercapturic acid (conjugate of acetyl-Cys) is excreted in urine mercapturic acid

42. C) Metallothioneins • Small proteins (~ 6,5 kDa), cysteine-rich  the –SH groups bind metal ions:Cu2+, Zn2+, Hg2+, Cd2+ • In cytosol, mainly of the liver, kidney, and intestine cells • Induced by metal ions • Functions: binding of metals, regulation of the Zn2+ level, transport of metals (Zn2+)