This document discusses the biotransformation of xenobiotics, or foreign compounds, in the body. Xenobiotics enter the body through food, medications, and industrial/pharmaceutical products. They are transformed in the liver and other tissues through two phases: Phase I involves reactions like hydroxylation and Phase II involves conjugating the compound to make it more polar and able to be excreted in urine or bile. Key enzymes involved in biotransformation include the cytochrome P450 system and conjugating enzymes that attach groups like glucuronic acid, sulfate, glycine, or glutathione to the xenobiotic. The liver is the primary site of biotransformation, which helps deactivate and eliminate toxins and drugs from the

1. Biotransformation
of xenobiotics
 Department of Biochemistry (J.D.) 2011
1

2. Greek word ξένος [xenos] means strange
• xenobiotics do not occur in the body
• they enter body mainly with food or as medications
• Chemical industry – produces synthetic compounds which
do not occur in nature (plastics, pesticides, dyes, additives…)
• Pharmaceutical industry – produces substances of synthetic
and natural (plant) origin – do not occur in the body
2

3. Entry of xenobiotics into body
• three principal entries: intestine, lungs, skin
• epithelium barrier between blood (ECF) and tissues (ICF) –
phospholipid bilayer
• penetration of xenobiotic depends on its physical and chemical
properties
• hydrophobicity facilitates the transport through cell membrane
3

4. Entry of xenobiotic into cells
• Simple diffusion – lipophilic substances, depends on concetration
gradient (liver – freely permeable, big pores in cell membrane, the
most affected in poisoning)
• Facilitated diffusion – transporters
• Active transport – primary, secondary
• Endocytosis
xenobiotics structurally similar with physiological
substrates can utilize all available transport systems
4

5. Biotransformation of xenobiotics in cells
• mostly in liver
• I. Phase – predominantly hydroxylations,
product may be still biologically active
• II. Phase – conjugation, product usually inactive
• products of biotransformations are more polar - they can be
excreted from the body by urine and/or bile
5

6. Excretion of xenobiotics from cell
• primary active transport – needs energy: ATP hydrolysis
• special ATP-ases called ABC (ATP binding cassettes)
• localized in cell membranes, export xenobiotics from cells into ECF
• MRP (multidrug resistence proteins) – in increased expresion, they
cause the resistance towards medicines
6

7. ABC = ATP binding cassettes
• superfamily of transmembrane proteins, they have ATP-binding
domain(s), substrate binding domain, and transmembrane domain(s)
• after ATP binding, ABC can translocate a chemical species across
membrane
• ABC are located in cell membranes as well as in intracellular membranes
• lipids, cholesterol, peptides, drugs, toxins etc.
7

8. Excretion of xenobiotics from body
• chemically modified (more polar) xenobiotics are excreted
by urine, bile  stool, or sweat
• volatile substance by lungs
• intestinal deconjugation and resorption sometimes occur -
enterohepatic circulation
• excretion into human milk
8

9. I. phase of biotransformation: examples of reactions
Reaction Xenobiotic (example)
Hydroxylation (P-450) (hetero)aromatic compounds (Ar-H  Ar-OH)
Sulfooxidation dialkylsulfide (R-S-R)  sulfoxide (R-SO-R)
Dehydrogenation alcohol / aldehyde hydrate  aldehyde / acid
Reduction nitrocompounds (R-NO2)  amines (R-NH2)
Hydrolysis ester  acid + alcohol
Reactions occur mainly in ER, some in cytosol
Enzymes of I. phase are rather non-specific – advantage !!
9

10. Cytochrome P450 (CYP)
• superfamily of heme enzymes (many isoforms)
• can catalyze different reaction types, mainly hydroxylation
• wide substrate specifity - advantage
• can be induced and inhibited
• occur in most tissues (except of muscles and erythrocytes)
• the highest amount in the liver (ER)
• exhibit genetic polymorphism ( atypical biotransformations)
Abbreviation: P = pigment, 450 = wave length (nm) of a absorption peak after
binding CO 10

11. Contributions of CYP isoforms to drug metabolism
4 % 1A2
1 % other
11 % 2C
52 % 3A4 30 % 2D6
2 % 2E1
11

12. Mechanism of CYP hydroxylation
• the formation of hydroxyl group
• monooxygenase: one O atom from O2 molecule is incorporated into
substrate between C and H (R-H  R-OH )
• the second O atom + 2H from NADPH+H+ give water
R-H + O2 + NADPH + H+  R-OH + H2O + NADP+
2 e- + 2 H+ 12

13. Components of cytochrome P450
+
2H
++
+ Fe
NADPH + H FAD hem
RH
O2
+ +++
NADP FADH 2 Fe
hem R OH H 2O
cyt. reduktasa cyt P-450
cytochrome P450 contains three cofactors and two enzymes:
• NADPH+H+, FAD, heme
• NADPH:CYP reductase (separates 2 H  2 e- + 2 H+) ER
• cytochrome P-450 (hydroxylase)
13

14. Detailed scheme shows reductive activation of O2
cyt P-450 A H
substrate A-H
substrát A H
3+
Fe
e
cyt P-450 cyt P-450 A H
NADPH + H
3+ 2+
Fe Fe
NADP
O2
e
2H cyt P-450 A H
2+
Fe O2
cyt P-450 A H
2+
Fe O2
A OH
hydroxylovaný
hydroxylated product
substrát
H2 O
14

15. Hydroxylation by CYP450 occurs
in endogenous and exogenous substrates
• Endoplasmic reticulum:
squalene, cholesterol, bile acids, calciol,
FA desaturation, prostaglandins, xenobiotics
• Mitochondria:
steroidal hormones
15

16. Compare various hydroxylations
Substrate Product Reagent Coreductant Other comp.
Phenylalanine tyrosine O2 BH4 * -
Xenobiotic xen-OH O2 NADPH+H+ FAD, heme
Proline 4-OH-Pro O2 2-oxoglutarate Fe2+, ascorbate
Dopamine noradrenaline O2 ascorbate Cu2+
* tetrahydrobiopterine
16

17. Main isoforms of human cytochrome P450
Various isoforms prefer different substrates, have different inducers and inhibitors
CYP Substratea Inducera Inhibitora
CYP1A2 theophylline tobacco smoke erythromycin
CYP2A6 methoxyflurane phenobarbital methoxsalem
CYP2C9 ibuprofen phenobarbital sulfaphenazole
CYP2C19 omeprazole phenobarbital teniposide
CYP2D6 codeine rifampicine quinidine
CYP2E1 halothane alcohol disulfiram
CYP3A4 diazepam phenobarbital grapefruit
a Examples from many possible compounds. 17

18. Inducers and inhibitors of CYP450
• some xenobiotics induce the synthesis of CYP – the metabolic
capacity of CYP is enhanced
• if administered inducer + drug, both metabolized by the same CYP
isoform  drug is metabolized faster  drug is less effective
• some xenobiotics inhibit CYP
• the most common isoform CYP3A4 metabolizes more than 120
different pharmaceutical drugs
• inhibitors of CYP3A4 are e.g. macrolide antibiotics, grapefruit
(furanocoumarins), ketoconazole
• if administered inhibitor + drug  increased drug level 
overdosing  side effects
18

19. Genetic polymorphism of CYP450
usual drug dose
(ultra)rapid
poor metabolizer extensive metabolizer
metabolizer
most of population
higher drug level normal response no / insufficient effect
side effects clinical effect of drug
intoxication
19

20. Example
I. Phase of biotransformation of benzene
hydroxylation
hydroxylace
(CYP 450)
H O
H
20

21. Example
Biotransformation of polycyclic aromatic
hydrocarbons (PAH)
H2O
HO
O
epoxid
reactive epoxide OH dihydrodiol
benzo[a]pyrene
O
interactions with DNA, mutations
vazba na DNA, mutace
tumours (kůže, lungs)
nádory (skin, plíce)
HO
OH
21

22. PAH in environment
• Industrial sources: combustion of fossil fuels (coal, petroleum oil,
etc.), production of coke, asphalt ...
• Non-industrial sources: forest fires, combustion of household
rubbish, cigarette smoke …
• Foods: fried, grilled, smoked, roasted foods, overheated fats and
oils, burnt (singed) bread, pastry …
22

23. II. Phase of biotransformation
• conjugation – catalyzed by transferases
• synthesis = endergonic reaction, one of the reactants must be
activated
• xenobiotic after I. phase reacts with endogenous conjugation
reagent
• conjugate is more polar, less active, easily excreted by urine
and/or bile (stool)
23

24. Conjugation reactions and reagents
Reaction Reagent Group in substrate
Glucuronidation UDP-glucuronate -OH, -COOH, -NH2
Sulfation PAPS -OH, -NH2, -SH
Methylation SAM -OH, -NH2
Acetylation acetyl-CoA -OH, -NH2
Sulfide formation glutathione Ar-halogen, Ar-epoxide
Amide formation glycine, taurine -COOH
24

25. Biosynthesis of UDP-glucuronate
P O CH2 HO CH2 HO CH2
O O UTP O
OH OH OH
HO OH HO O P HO O UDP
OH OH OH
glukosa-1-P UDP-glukosa
UDP-glucose
glukosa-6-P
glucose 6-P glucose 1-P
+
O O NAD
C H2O
+
O NAD
OH
glukosiduronáty
(bis)glucosiduronates HO O UDP
OH
UDP-glukuronát
UDP-glucuronate
25

26. UDP-glucuronate
COO
O
OH O
HO O
O NH
HO P O
O
O P N O
O O
O-glycoside bond O N-glycoside bond
of ester type
OH OH
26

27. Glucuronates are the most common conjugates
• O-glycosides
ether type (Ar-O-glucuronate, R-O-glucuronate)
ester type (Ar-COO-glucuronate)
• N-, S-glycosides
• Substrates: aromatic amines, amphetamines,
(acetyl)salicylic acid, drugs, flavonoids ...
• Endogenous substrates: bilirubin, steroids
27

28. bilirubin bisglucosiduronate
28

29. Example
Biotransformation of amphetamine
amphetamine
Phase I reaction
Phase II reaction
4-hydroxyamphetamine
4-hydroxyamphetamine
4-O-glucosiduronate
Phase I reaction
Phase II reaction
4-hydroxynorephedrine
4-hydroxynorephedrine
4-O-glucosiduronate 29

30. PAPS phosphoadenosyl phosphosulfate
NH 2
Physiological sulfations: N
O N
O
Glycosaminoglycanes S O
O N N
heparine, dermatane sulfate, O P
keratane sulfate, O
O O
chondroitine sulfate etc.
Sulfoglycosphingolipids
O O OH
(acidic glycolipids, sulfatides)
P
O
O
30

31. Example
Biotransformation of phenol
hydroxylation
hydroxylace
(CYP 450)
H OH
conjugation
konjugace
O glucuronate
glukuronát O SO3H
sulfát
31

32. Glutathione – three functions
-glutamyl-cysteinyl-glycine
• Reductant = antioxidant (glutathione peroxidase)
• Conjugation agent (glutathione transferase)
endogenous substrates – leukotrienes
• AA Transport into cells (-glutamyltransferase, GMT)
32

33. Glutathione (GSH)
NH2 H O

N
HOOC  N COOH

O CH2 H
SH
electrophilic
site
R-X + GSH  R-SG + XH (R-X epoxides, halogenalkanes)
nucleophilic
group
33

34. Example
R-SG sulfide is converted to mercapturic acids
and excreted
CoA-SH
acetyl-S-CoA
GSH
Glu + Gly
epoxide
N-acetyl-S-substituted cysteine
(mercapturic acid)
34

35. Methylations are involved in the inactivation of catecholamines
MAO monoamine oxidase, COMT catechol-O-methyltransferase
MAO O
HO CH2 CH2 NH2 HO CH2 C
- NH3 H
dopamine
HO HO
dihydroxyphenylacetaldehyde
O O
COMT
HO CH 2 C
HO CH 2 C
OH
SAM OH
O
HO
CH 3 homovanillic acid dihydroxyphenylacetic acid
Inactivation can proceed in the reverse order: first COMT, then MAO, product is the same. 35

36. Conjugation with amino acids
(amide formation)
• glycine, taurine
• xenobiotics with -COOH groups
• amide bond formation
• endogenous example: conjugated bile acids
36

37. Toluene biotransformation
CH3 CH2OH COOH
toluene benzylalcohol benzoic acid
O glycine
glycin O
C C O
OH NH CH2 C
OH
benzoic acid
benzoová kys. hippurová kyselina
hippuric acid
(activated by CoA) (N-benzoylglycin)
(N-benzoylglycine)
37

38. 38

39. Biotransformation of ethanol in liver (cytosol)
H H alkoholdehydrogenasa
alcohol dehydrogenase (AD)
O
H3C C O + NAD H3C C + NADH+ H
H H
acetaldehyd
acetaldehyde
aldehyddehydrogenasa
acetaldehyde dehydrogenase (AcD)
H H H
NAD O
H3C C O + H2O H3C C O H3C C
- 2H
 NADH + H+
OH OH
aldehyd-hydrát
acetaldehyde hydrate acetic acid
octová kyselina
39

40. • Alcohol dehydrogenase (AD) – metalloenzyme (Zn), more
isoforms, in liver, lungs, kidneys, intestine, and other tissues
• some isoforms are less active in females
• Acetaldehyde dehydogenase (AcD) – more isoforms, liver,
cytosol and mitochondria
40

41. Alternative pathways for alcohol metabolism
ER:
MEOS (microsomal ethanol oxidizing system, CYP2E1)
CH3-CH2-OH + O2 + NADPH+H+  CH3-CH=O + 2 H2O + NADP+
It is activated on higher alcohol levels (> 0.5 ‰)  increased
production of acetaldehyde
Peroxisomes: oxidation of ethanol by hydrogen peroxide, catalase
CH3-CH2-OH + H2O2  CH3-CH=O + 2 H2O
41

42. Metabolic consequences of EtOH biotransformation
Ethanol
AD, AcD
AD
MEOS
part. soluble acetaldehyde excess of NADH in cytosol
in membrane PL (hangover)
reoxidation by pyruvate
adducts with acetate
proteins excess of lactate  acidosis
nucleic acids
toxic effects amines lack of pyruvate  hypoglycaemia
on CNS acetyl-CoA
various products
FA/TAG synth.
liver steatosis 42

43. Acetaldehyde reacts with biogenic amines
to tetrahydroisoquinoline derivatives (animal alkaloids)
HO HO
NH2 N
HO - H2O HO H
dopamine
CH 3
H O
C
salsolinol
CH3
6,7-dihydroxy-1-methyl-1,2,3,4-
acetaldehyde tetrahydroisoquinoline
Neurotoxin ?
43

44. Tests for detection of ethanol intake
Liver enzymes: GTM, AST, ALT, GMD, CHS
Fatty acids ethyl esters (FAEE) appear in the blood in 12 – 18 h after drinking and can be
detected even 24 h after alcohol in blood is no more increased. However, traces of FAEEs are
deposited in hair for months and may serve as a measure of alcohol intake.
Ethyl glucosiduronate (EtG) increases in the blood synchronously with the decrease of
blood ethanol and can be detected (in the urine, too) after few days, even up to 5 days.
Phosphatidyl ethanol (PEth) is present in the blood of individuals, who have been drinking
moderate ethanol doses daily, in even 3 weeks after the last drink.
Carbohydrate-deficient transferrin (CDT). In the saccharidic component of each
transferrin molecules, there are 4 – 6 molecules of sialic acid. Drinking to excess disturbes the
process of transferrin glycosylation so that less sialylated forms of transferrin (with only two
or less sialyl residues per molecule, CDT) are detected in blood during approximately 4
weeks after substantial alcohol intake.
44

45. Per milles of alcohol in blood
‰ = per mille = 1/1000
malcohol (g)
alcohol in blood (‰) =
mbody (kg)  f
Biological feature Males Females 0.67 (males)
Total body water 60 – 67 % 50 - 55 % 0.55 (females)
Total body fat 10 – 20 % 20 – 30 %
45

46. Oxidation of ethylene glycol proceeds stepwise
with a number of intermediates
CHO
oxid. oxid.
CH2OH CHO CHO COOH COOH
oxid. oxid.
glyoxal
CH2OH CH2OH oxid. oxid. CHO COOH
ethylenglykol
ethylene glykolaldehyd
glycolaldehyde glyoxalic
glyoxalová št'avelová
oxalic acid
COOH kyselina
kyselina
acid
glycol
CH2OH
glykolová kyselina
glycolic acid
in kidneys  calcium oxalate stones  renal failure
46

47. Tobacco
Substances
nicotine, the products of incomplete combustion
involved
euphoria, psychical relaxation, increase of pulse rate,
vasoconstriction, stimulates adrenaline release (silent stress),
Effects
increases salivary and gastric secretion, stimulates intestinal
peristalsis (defecating effect of the first morning cigarette)
Symptoms
typical smell, yellow fingers and teeth
of abuse
lung diseases (COPD*, cancer), heart attack,
Risks
erectile dysfunctions, premature wrinkles
* chronic obstructive pulmonary disease 47

48. Nicotine is the principal alkaloid of tobacco
2
3 more basic
1 N pKB = 6,16
2 CH3
N
1
less basic
pKB = 10,96
3-(1-methylpyrrolidine-2-yl)pyridine
48

49. What happens during cigarette burning?
• temperature about 900 C
• dried tobacco undergoes incomplete combustion
• very complicated mixture of products
• nicotine partly passes to smoke, partly decomposes
Cigarette box
Nicotine: 0.9 mg/cig.
Tar: 11 mg/cig.
49

50. Cigarette smoke contains
• free base of nicotine – binds to receptors in the brain
• CO – binds to hemoglobin to give carbonylhemoglobin (tissue ischemia)
• nitrogen oxides – may generate reactive radical species
• polycyclic aromatic hydrocarbons (PAH)
(pyrene, chrysene, benzo[a]pyrene …), main components of tar
they can attack and damage DNA, carcinogens
• other substances (N2, CO2, HCN, CH4, esters …)
50

51. How to disclose a smoker?
1. saliva test
smoker’s saliva contains much higher level of thiocyanate
than saliva of non-smoker,
thiocyanate is generated from CN- → SCN-
reaction with Fe3+ ions give red complex
2. nicotine in urine
3. minor tobacco alkaloids in urine
(cotinine, nornicotine, anatabine, anabasine)
51

52. Example
Biotransformation of nicotine
N
CH3
N
nicotine
N N OH
H CH3
N N
nornicotine 5-hydroxynicotine
nicotine-N-glucuronate
N O
cotinine-N-glucuronate CH3
N
cotinine
52

53. Biotransformations of selected drugs
Drug Biotransformation Metabolite
Codeine demethylation morphine (active, another way)
Bromhexin hydroxylation + demethylation ambroxol (active, the same)
Paracetamol conjugation, oxidation conjugates (mostly inactive)
Aspirin hydrolysis, hydroxyl., conjug. conjugates (inactive)
53

54. Bromohexin is the prodrug of an expectorant ambroxol
N-demethylation
hydroxylation
bromohexin ambroxol
(prodrug) (expectorant)
Antitussic codeine (3-O-methylmorphine) is transformed slowly into morphine
O-demethylation
codeine morphine
(antitussic) (analgesic, an addictive drug)
54

55. Acetaminophen (p-acetaminophenol, paracetamol)
N-(4-hydroxyphenyl)acetamide
prepared in 1893, common analgetic-antipyretic,
overt the counter, without a prescription
The amide bond is not hydrolyzed!
oxidation of only a small part to
cyt P450 N-acetyl-p-benzoquinoneimide (NAPQI),
unless the conjugating capacity is exhausted
~ 3 % excreted
unchanged
into the urine if conjugation capacity
CONJUGATION is limited,
GSH unwanted side effects:
– covalent bonding
to proteins,
– oxidation of –SH groups
in enzymes,
– depletion of GSH,
– hepatotoxicity at
60 % as glucosiduronate overdosing
30 % as sulfate ester mercapturic acid
55

56. Acetylsalicylic acid (Aspirin)
is an analgetic-antipyretic with antiinflammatory effect; over the counter,
minute doses inhibit aggregation of blood platelets.
acetylation of macromolecules
(acetylation of COX inhibits
the synthesis of prostaglandins)
esterase
UDP-glucuronate
and O
UDP salicyl glucosiduronate salicyloyl glucosiduronate
salicylate
cyt P450
glycine
o-hydroxyhippurate
(salicyloylglycine,
salicyluric acid)
gentisate oxid.
2,5-dihydroxyhippurate
quinone (gentisoylglycine,
(and products of its glycine gentisuric acid)
polymerization)
56

57. Polypragmasy - application of multiple remedies simultaneously
• it is proper to avoid application of too many different remedies together
• interactions between different drugs or their metabolites can cause
enhancement or inhibition of pharmacological effects
• the mixed type hydroxylases (cyt P450) are inducible, their activities
may increase many times in several days, so that the remedies are less efficient
• if the load of the detoxifying system is high, minor pathways of transformation can be
utilized and produce unwanted side-effects due to the formation of toxic metabolites
• intensive conjugation with glutathione can result in depletion of this important
reductant in the cells
57

58. Selected biochemical markers of liver damage (in serum)
Analyte Reference values Change
ALT 0,1 - 0,8 kat/l 
GMD 0,1 - 0,7 kat/l 
GMT 0,1 - 0,7 kat/l 
Bilirubin 5 - 20 mol/l 
Ammonia 5 - 50 mol/l 
Urobilinogens (urine) up to 17 mol/l 
------------------------------ --------------------- -------------
Pseudocholinesterase 65 - 200 kat/l 
Urea 3 - 8 mmol/l 
Albumin 35 - 53 g/l 
58