Disposition: Biotransformation (II)

glucuronidation conjugation reaction

• enzymatic mediator: UDP-glucuronosyltransferase (micorosomal membrane-bound, transfers glucuronate group of uridine diphosphoglucuronate to functional group) (two families, 3 subfamilies)

• location: ER

• endogenous substrate: steroids, thyroxine, catacholamines, bilirubin

• functional groups: -OH, -COOH, -NH2, -NH, -SH, -CH

• major phase II pathway, 35% of drug conjugation

• UDPGA synthesized from glucose-1-phosphate, which is present in high concentrations in cells

• UDP and G1P form USP-glucose, dehydrogenation forms UDPGA

• UDP-glucuronate + acceptor → UDP + acceptor-beta-D-glucuronide

• results in highly polar, water soluble molecule (most effective for water solubility)

• resulting molecule excreted via urine or bile

• xenobiotics conjugated with glucuronic acid can be substrates for beta-glucuronidase, which can release the parent or phase I metabolite to be reabsorbed (enterohepatic circulation)

  • delays elimination of xenobiotic

• typically reacts with nucleophiles

sulfate conjugation reaction

• enzymatic mediator: sulphotransferase (SULT1, SULT2,)

• location: cytosol

• endogenous substrate: steroids, carbohydrates

• functional groups: aromatic -OH, aromatic -NH2, alcohols

• transfer of a sulfate group from 3-phosphoadenosine 5-phosphosulfate (PAPS) to hydroxyl group on acceptor

• increases water solubility

• >11 enzymes, 3 active as dimers with broad substrate specificities

• some SULTs in Golgi for sulfonation of proteoglycans

• typically reacts with nucleophiles

SULT enzyme classes

• arylsulfotransferase: sulfonates phenolic xenobiotics (SULT1)

• alcohol sulfotransferase: primary and secondary alcohols (SULT2)

• estrogen sulfotransferase: estrogen and aromatic hydroxysteroids (SULT1)

• tyrosine ester sulfotransferase: tyrosine methyl ester and 2-cyanoethyl-N-hydroxythioacetamide

• bile salt sulfotransferase: conjugated and unconugated bile acids (SULT2)

glutathione conjugation reaction

• enzymatic mediator: glutathione S-transferase (GSTs)

• location: cytosol & ER

• endogenous substrate: metabolites of arachidonic acid, epoxides, alkenes or electrophilic C centers, sulfur or nitrogen centers

• functional groups: epoxide, organic halides

• GSTs have five classes (alpha, mu, pi, theta, and zeta), can be microsomal (encoded by single genes), can be homo- or hetero-dimeric

• typically reacts with electrophiles

• excretion via bile and conversion to cysteine and mercapturic acid conjugates in intestine and kidneys

• can activate compunds to reactive species (haloalkanes)

• displacement reaction: GTH replaces electron-withdrawing group

• addition reaction: GTH addted to activated double bond or strained ring

amino acid conjugation reaction

• enzymatic mediator: aminotransferase

• location:

• endogenous substrate: benzoic acid, phenylacetic acid, alicylic acid

• functional groups: aromatic -NH2, -COOH

• carboxyl group reacts with amino group

  • amino group of acids: glycine, taurine, glutamine

  • carboxyl group of acids: proline, serine

• xenobiotics with carboxylate can be activated with coenzyme A to produce acyl-CoA thioestes then conjugated (prevents deprotonation of carboxyl)

phenylacetic acid

treats hyperammonemia (excessive ammonia in bloodstream)

acetylation conjugation reaction

• enzymatic mediator: acetyltransferase (also use acetyl CoA)

• location: cytosol

• endogenous substrate: seratonin

• functional groups: aromatic -NH2 (preferred), aliphatic -NH2, hydrazines (preferred), -SO2HN2

• acetyl CoA + aryl amine → CoA + N-acetlyarylamine

• ping-pong Bi-Bi mechanism:

  • acetyl group transferred from acetyl-CoA to cysteine in NAT, release of coenzyme A

  • acetyl group released from NAT to substrate, regeneration of enzyme

methylation conjugation reaction

• enzymatic mediator: methyltransferase (cofactor is S-adenosylmethionine, most widely used after ATP)

• location: cytosol & ER

• endogenous substrate: biogenic amines

• functional groups: aromatic -OH, -NH2, -NH, -SH

• common but minor pathway; does not increase water solubility

• MTs are small, cytosolic, monomeric enzymes that utilize SAM as methyl donor

exceptions to the rule

acetylation (single oxygen atom) and methylation (no electronegative atoms) do not produce more water soluble compounds (only detoxify, do not facilitate excretion)

impact of species on biotransformation

• differences in species capability to biotransform specific chemicals is basis for selective toxicity

  • mammals can biotransform malathion to safe metabolites, insects oxidize to lethal metabolite

• humans have higher capacity for glutamine conjugation than lab rats

  • most other enzymes and biotransforming reactions are comparable

impact of gender on biotransformation

• usually limited to hormone-related differences

• cocaine: males metabolize 2x faster

• diazepam: males metabolize faster

• hexobarbital and pentobarbital: males metabolize faster (females get higher blood leves and prolonged sleep time)

• indinavir: males metabolize 3x faster

• morphine: males metabolize faster

• tolbutamide: males metabolize faster

• acetominophen: lower glucuronidation in females results in higher parent plasma concentration

• aspirin: higher esterase activity in males results in lower plasma levels

• nortriptyline: higher metabolism in males results in lower plasma levels

• propanolo: lower glucuronidation in famales results in higher urinary excretion of parent compound

impact of age on biotransformation

• fetuses and neonates have limited ability for xenobiotic biotransformations

  • cholinesterase enzyme system more vulnerable to inactivation (greater susceptibility to organophosphate pesticides)

  • Cyp1A2, Cyp2C9, Cyp2C19 not fully operational in early infancy

  • Cyp2E1 not fully operational until 6-12 months

  • many phase II enzymes not functional in newborn period

  • xenobiotic half-life can be 3-9x longer compared to adults

• capacity for biotransformation fluctuates with age in adolescence but stabilizes by early adulthood

• capabilty decreased in elderly

impact of nutrition on biotransformation

• inadequate levels of proteins, vitamins, and essential metals can decrease ability to synthesize biotransforming eznymes or limit co-factor supply

impact of disease on biotransformation

can impar capacity to biotransform xenobiotics

impact of genetic variability on biotransformation

• largest variation in biotransformation capability among humans, particularly for phase II

• with slow acetylators, acetylation is so slow that blood or tissue levels of drugs or phase I metabolites exceed toxic threshold

impact of enzyme inhibition or induction on biotransformation

• caused by prior or simultaneous exposure to xenobiotics

• major mechanism is competition between substances for oxidizing or conjugating enzymes

• most commonly induced enzyme reactions are the CYPs

impact of dose level on biotransformation

• biotransformation may be different at high doses than low, contributes to dose threshold

• mechanism explained by different biotransformation pathways

  • low doses may follow pathway leading to detoxification

  • saturation may cause parent toxin to build up or entry of a different biotransformation pathway

Acetominophen biotransformation

• at normal doses, ~96% is biotransformed to non-toxic metabolite by sulfate and glucuronide conjugation, 4% conjugated to toxic metabolite (detoxified by glutathione)

• 7-10x recommended dose, conjugation pathways are saturated; high toxic concentration and GTH in liver depleted preventing detoxification

• metabolite reacts with liver proteins and causes fatal liver damage