Transcript Detoxikace endogenních a exogenních látek

Detoxification of endogenous and
exogenous compounds
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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)
Nitrogen removal from amino acids
transamination
oxidative
deamination
urea cycle
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
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:
L-alanine
pyruvate
-ketoglutarate
glutamate
L-aspartate
oxalacetate

Transamination is catalyzed by transaminases (aminotransferases) that require participation of pyridoxalphosphate:
amino acid
pyridoxalphosphate
Schiff base
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
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 deaminated releasing ammonia that
– in the liver – enters the urea cycle, or used for syntheses
 Aspartate enters the urea cycle
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
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 liver and kidney and deaminated by
L-glutaminase:
Amide nitrogen, not the
-amino nitrogen is removed!

Glu can be oxidatively deaminated, ammonia is excreted by the
kidneys or converted to urea in the liver
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 of pH of the urine
Transport of nitrogen – 2) as Ala

Mainly by the muscle

i.a. in the glucose-alanine cycle:
Liver
Muscle

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):
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
Detoxification of ammonia

a) ammonia is built into Glu by glutamate dehydrogenase and
Gln 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
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:
serine dehydratase
– NH3
By analogy: Thr to α-ketobutyrate

Bacteria in the gut also produce ammonia
Urea cycle

In the liver in 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 +
carbamoyl phosphate


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
Fumarate
← Glu + oxalacetate

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
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
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:
 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
Other nitrogenous degradation
products excreted in the urine

Creatinine – produced from creatine phosphate:

Uric acid – degradation product of the purine bases
B) Metabolism of xenobiotics

Drugs, preservatives, pigments, pesticides …

Predominantly in the liver, also in the intestines, kidney, lungs

Involves two phases
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
Potential toxic effects of activated
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 1 can 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
Reactions of phase 1:

Hydroxylation

Epoxide formation

Reduction of carbonyl-, azo-, or nitro- compounds

Dehalogenation
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 NADPHcytochrome P450 reductase, then to cytochrome P450 and
to oxygen → one oxygen atom is inserted into the substrate
 They metabolize not only xenobiotics but also endogenous
compounds, e.g. some steroids, eicosanoids
Isoforms of cytochrome P450

Hemoproteins in the endopl. reticulum, inner mitoch. membrane

Most abundant in the liver and small intestine followed by lungs

Nomenclature based on the AA sequence identity:
CYP3A4
CYP = cytochrom P450
3…family
4…isoform number within the subfamily
A…subfamily

Some exist in polymorphic forms, some of which exhibit low
activity  accumulation of the corresponding xenobiotic

Some are involved in metabolism of polycyclic aromatic hydrocarbons (PAHs), thus playing a role in carcinogenesis
Most isoforms are inducible:

E.g. by phenobarbital and other drugs, but also by their own
substrates

Mechanism: mostly increased transcription

Can lead to drug 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
Metabolism of ethanol
– mainly in the liver
Most of acetate enters the blood and, mainly in the skeletal muscle, is activated to acetyl-CoA → TCA cycle

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 also some
carcinogenic components of tobacco smoke!
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)
Glucuronidation

UDP-glucuronic acid is the glucuronate donor:
glucuronate

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
Bilirubin excretion

Bilirubin is the product of heme catabolism:
heme
M: methyl, V: vinyl,
CE: carboxyethyl (propionic)
transported to the liver bound
to albumin
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 reexcreted through the
liver into the bile
a small fraction is
excreted into the
urine by the kidney
most of them are oxidized to pigments and
excreted in the faeces
(urobilin, stercobilin)
Sulfation

Some alcohols, arylamines, phenols, but also glycolipids, steroids

Sulfate donor: PAPS (3´-phosphoadenosine-5´-phosphosulfate):
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!
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
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+)