Lecture 6/7 - Types of Phase II Biotransformation

Question 1

As we age xenobiotic elimination is decreased so is GFR correlative to elimination rate and to what age is this valid?

Answer 1

how fast can your kidneys eliminate - glomerular filtration rate be correlative to elimination rate - this is how much blood and soluble solutes from xenobiotics get pushed out of body - normal GFR is anything above 100
-measure GFR past 80 and do the same math and GFR is 10 what does that mean
140-80(age) * weight in kg (40) /72(1.4) * 0.85 = 23.2mL/min
normal even at that age is above 60 so something id wrong with the GFR calculation - this equation only goes up to 70 years old so can’t use equation so you can’t extrapolate never experimentally tested
-kidney failure people can’t eat or move
-above 60 at age 80 years old is Olay for GFR
-urine is actual test and and expel it over time to expel the creatinine - breakdown product of muscle

Question 2

How long does food stay in stomach? Where does it go from there and where are carbs broken down?

Answer 2

like 30minutes then goes to the small intestine which are lipases
-carbs broken down form starch into sugar and this is broken down by acids

Question 3

Why can there be an energy requirement for phase ii metabolism?

Answer 3

it is needed for homeostasis because there are transporters there to excrete the phase ii conjugation with polar group
-this depends on abc transporter (atp binding cassettes) - ABCC1, C2, C3, C4 - want to know if actively transported and gave a drug and it binds to these then get accumulation and can get ddi; body sees aldehydes and carbonyls as potential harm - in general if conjugated it is active transporter so if anything is conjugated it is actively transported

Question 4

Why are you less likely to get colon cancer with more bowel movements?

Answer 4

because you are not reabsorbing cause of inflammation

Question 5

According to the fda what is the classification for anything conjugated?

Answer 5

anything conjugated is nontoxic

Question 6

Where does phase ii metabolism occur and what are the products of phase ii metabolism and what conjugation is included?

Answer 6

-cytosol of cell
-products are generally water soluble and easily excreted
-sugar conjugation, sulfation, methylation , acetylation, amino acid conjugation, glutathione conjugation

Question 7

What do schiff base conjugations of amino acids cause?

Answer 7

Hoping that Schiff base conjugations of amino acid conjugations get cleared and some can stay around and cause immunological response

Question 8

What product from the breakdown of ethanol burns the liver?

Answer 8

formic acid

Question 9

Do hormones like testosterone and estrogen have toxic metabolites?

Answer 9

yes

Question 10

What are some phase ii reactions?

Answer 10

glucuronic acid conjugation
sulfate conjugation
glycine, glutamine, and other amino acid conjugation
glutathione conjugation
mercapturic acid synthesis
acetylation
methylation

Question 11

Why are epoxides toxic?

Answer 11

they are toxic because they cause steric hindrance and they form an aldehyde and then the aldehyde conjugates into glutathione

Question 12

What does a diet high in sulfites like broccoli and cauliflower prevent?

Answer 12

allows for sulfation and prevents prostate cancer

Question 13

Nucleophiles undergo the sulfating acetylation and glucoronidation?

Answer 13

-most toxic gets glucoronidated
-important to take medicine like Tylenol with food cause need glucose with glucuronidation

Question 14

What is bilirubin?

Answer 14

end product of heme degradation

Question 15

What happens every 120 days when red blood cells die?

Answer 15

so heme to biliverdin and then bilirubin and then bile acids - the heme is broken down and recycled

Question 16

What is the majority of bilirubin derived from?

Answer 16

the break down of senescent erythrocytes

Question 17

What is heme oxygenized to and then what is that product reduced to and what cells do this?

Answer 17

heme is oxygenized to biliverdin and reduced to bilirubin by phagocytes

Question 18

What is the solubility of bilirubin and is bound to what to be transported to the liver?

Answer 18

water insoluble, bound to albumin and transported to the liver

Question 19

Upon uptake into the liver what happens to bilirubin?

Answer 19

it will be conjugated with one or two molecules of glucuronic acid by bilirubin UDP-glucuronyl transferase in the ER
-conjugated bilirubin is non toxic and water soluble and is excreted into the bile
-most bilirubin glucuornides are deconjugated in the intestines to urobilinogen and 20% is reabrsorbed and primptly excreted via the bile

Question 20

What happens to the globulin of hemoglobin?

Answer 20

it undergoes proteolysis

Question 21

What does conjugation with UDPGA via the SN2 reaction require?

Answer 21

a nucleophile or handle on the drug or xenobiotic

Question 22

What are some targets for glucuronidation?

Answer 22

hydroxyl group
sulfuryl group
primary amine

Question 23

When does enterohepatic recirculation occur and what is it?

Answer 23

-2-6 hours after circulation
-Have bacteria in go tract and can break this off and in large intestine can reabsorb

Question 24

What is the most widespread, important of the conjugation reactions?

Answer 24

UGTs and the glucuronidation reaction
-cofactor udp-glucuronic acid is high in abundance
-closely related to glycogen synthesis and found in all tissues of the body
-other sugars glucose xylose or ribose may be conjugated

Question 25

What does the microsomal enzyme glucuronyl transferase do?

Answer 25

conducts the donation of glucuornic acid from the endogenously - synthesized UDPGA to various substrates to form glucuronide conjugates

Question 26

What are some glucuronides formed?

Answer 26

RN-G, RO-G, RCOO-G, RS-G, RC-G

Question 27

What is an endogenous substrate of glucuronides?

Answer 27

bilirubin

Question 28

What is glucuronide expression induced to?

Answer 28

phenobarbital

Question 29

What is CPT11 metabolism and the role of udp glucuronyl transferase in regards to polymorphisms of UGT 1a1?

Answer 29

-associated with homoygoisty for a dinucleotide insertion TA into the TA6TAA elements in the UGT1A1 promoter a0dditional TA in the promoter reduces transcirption of the enzyme -UGT1A1 decreased activity in hypoglycemic or malnourished conditions -7 ta repeats reduced rate of transcription protein and glucuornidation of UGT1A1 substrates -reduced 30-50% bilirubin glucuronidation = gilberts syndrome -UGT1A1*28 associated with increased toxicity with irinotecan

Question 30

What is the effect of conjugation on directing metabolite excretion?

Answer 30

-there is a qualitative increase in biliary excretion of compounds with a higher molecular weight and thus conjugation to metabolites that are more polar nad ionic than the parent drug enhances the bile/plasma ratio of a metaboliterelative to the parent drug -threshold of 400-500 dlatons for favorable biliary vs renal excretion of small organic molecules

Question 31

How are conjugated drugs eliminated out of cells?

Answer 31

they can be into the bile or urine using transporter related excretion -drugs get pumped out and goes in the liver

Question 32

How is the microbiome another metabolic organ?

Answer 32

This phase ii gets deoncjugation due to bacteria so the microbiome is another metabolic organ

Question 33

Where are glucuornides excreted?

Answer 33

into the bile, plasma, urine

Question 34

What are some potential dfferenecer in the lucuornide metabolited formed?

Answer 34

stability of the glucuronide product can lead to chemical hydrolysis free sn38 found In gi tract causes diarrhea so bacteria in gi tract causes that problem

Question 35

What are some drug mediated toxicites?

Answer 35

Metabolism can form AGEs - glycosylation of proteins and this is like hemoglobin a1c and these sugar molecule binds on to drugs and cause immunological reactions -protein bound to drug and then drug gets glycosylated causing immunological response -essentially a protein binds to a drugs and gets glycosylated and this causes an immunological response

Question 36

What are the two major human families of UGT?

Answer 36

UGT1 and UGT2 -do these UGT isoforms have different roles

Question 37

What is the biochemical pathway for cosubstrate UDP-GA?

Answer 37

ATP + UDP ---> UTP + ADP ATP + Glucose ----> Glycogen + Glucose 1-P UTP + Glucose-1-P ---> UDP-glucose + PP UDP-glucose + NAD+ ---> UDP-glucuronic acid + NADH UDP-glucuronic acid + ROH ----> RO-glucuornide + UDP

Question 38

Where does glucose-1-P come from?

Answer 38

glycogen and the reactions are rapid so co substrate depletion is not often observed unless in starved animals

Question 39

In what cases is co substrate depeltion observed?

Answer 39

in animals with large doses of drugs but recover of UDPGA is rapid and not likey to occur in humans unless there is a serve overdose which occurs

Question 40

What is the rate limiting step in the biochemical pathways for cosubstrate UDP-GA?

Answer 40

transport from the cytosol to the ER in vitro

Question 41

What compounds can depleye UDP-GA through other mechanisms?

Answer 41

diethyl ether halothane and phenobabrital

Question 42

What type of enzyme is UGT and what influences the rate of the enzyme

Answer 42

UDP-GA influences the rate and UGT is a bi substrate enzyme - inactivated microsomes in vitro is 25-40mM and 2-10mM with activation

Question 43

What are some methods for characterizing metabolites as glucuronides?

Answer 43

susceptibility to beta glucudornidase -sources of beta glucuornidase - controls - 1,4-saccharolactone - positive controls -release of glucuronic acid is a reducing sugar by beta glucurnidase -spetroscopy using nmr and ms

Question 44

How can mass spec be used to id and quantify glucuronides?

Answer 44

mass spec provides M+H+ and loss of sugar 176 which can also occur via in source fragmentation -no information on the site of glucuronic attachment or isomerization (acyl migration) is provided via MS -site of attachment may be possible if other fragmentations of drug occur with retention of sugar

Question 45

Do glucuornides always result in less toxicity or inactivation?

Answer 45

no - but most are inactive -morphine glucuronide is pharm actuve and acyl glucuornides are reactive and they can bind covalently to proteins -acetylaminofluorene hydroxylamine glucuornide is reactive

Question 46

Would one expect M6G to be absorbed orally?

Answer 46

morphine-6-glucuronide (M6G) actually can be absorbed orally, but its absorption is limited. M6G is a metabolite of morphine that has significant analgesic properties, and while it has low oral bioavailability compared to morphine itself, it still can be absorbed to some extent through the gastrointestinal tract. The main issue is that M6G undergoes extensive first-pass metabolism in the liver, leading to reduced bioavailability. However, it is still found in the plasma when morphine is taken orally, albeit at lower levels than morphine itself. So, while M6G can be absorbed orally, it’s not absorbed efficiently compared to morphine, and its effects are more prominent when morphine is administered parenterally (e.g., IV).

Question 47

Would M6G have the same tissue distribution as morphine?

Answer 47

M6G, however, has a much lower ability to cross the blood-brain barrier compared to morphine. This is due to its higher polarity and glucuronide conjugation, which makes it less lipid-soluble. As a result, M6G tends to be more confined to peripheral tissues and has more prominent effects on peripheral analgesia rather than CNS-mediated effects.

Question 48

What are some properties of clucuronide conjugates?

Answer 48

increase in MW of the product by 176 glucuronic acid is achiral so products are racemate adding glucuronic acid changes the metabolite to the parent drug an dincreases polairy affecting membrane transport tissue distribution

Question 49

What efflux transporters will efflux glucuornide?

Answer 49

MRP, OATP, BCRP, OAT will efflux glucuronides

Question 50

What are unstable and reactive?

Answer 50

acyl glucuronides -this is due to intramolecular acyl migration - transesterification -nuclophillic siplacement with aminoacid sidechains of proteins

Question 51

What happens if you glycosylate Hb or a protein?

Answer 51

Glycosylate Hb or a protein that is an antigen or a vaccine and protein should have an immunogen - vaccine mimics virus - post acute covid syndrome or long covid and this is autoimmunity we make autoantibodies against this and get rid of these gets PARS away

Question 52

What leads to severe disease or elevated bilirubin?

Answer 52

deificent conjugation with bilirubin -crigler najar or sever disease is associated with a 13 bp deletion in exon 2 of UGT1*1 -gilberts syndrome is associated with a defect in the promoter region of exon 1

Question 53

Why does gilberts syndrome lead to sevre neutropenia?

Answer 53

odds ratio of getting severe neutropenia - if they get CPT11 they cannot glucoronite sn38 - if you have GS you block this and SN38 gets increase of myelosuppresion or neutropenia - neutrophils engulf pathogens and fight infections - fight bacterial infections - neutropenia chances of dying

Question 54

What do variants of UGT1A9 appear to have?

Answer 54

they appear to create a fast metabolizer phenotype in 15% patients

Question 55

What is the UGT1A1*28 polymorphism associated with?

Answer 55

decreased glucuronidation of sn-38 higher levels of sn-38 higher grades of neutropenia and diarrhea

Question 56

What is UDP glucuronyl transferase 1a1 responsible for?

Answer 56

gilberts bilirubinemia - absent in 15% of caucasians - in less than 5% of asians and more than 50% of africancs and hispanics -decreased activity in hypoglycemia or malnoursihed conditions -only way to stop neutropenia is to stop chemo and then tumor comes back and then gcsf increases neutrophils, cause too many but that increases relapse and neptosis incurs and inflammation occurs and tumor comes back

Question 57

How can active metabolites be assessed?

Answer 57

structural identification of actuve metabolites -MS indicated presence fo monohydroxylation -NMR shows site of hydroyxlation subsequent steps -monohydroxylated metabolite synthesized -activity and pk properties confirmed

Question 58

What are some issues which can arise from reactive metabolites?

Answer 58

metabolic activation to reactuve intermediated and interfernece with metabolic processes -cellular hepatic and necrosis -idiosyncratic toxicity glutathione adducts protein covalent binding and immunogenic response DDI mechanism deprndent cyp inhibition

Question 59

What can glucuornic acid also conjugate to?

Answer 59

phenols, tertiary amine, aromatic amines

Question 60

What is the defintiion of a conjugation reaction?

Answer 60

conjugation is the addition of a moleclue to the drug or xenobiotic -glucuornidation -gsh (glutathione tripeptide derivative) sulfate, amino acid, conjugation acetylation methylation -Most cns drugs are glucoronidated because they are fatty - lower log p are fatty

Question 61

What is the increase in MW which each of the following conjuagtions: glucuronidation glycine - sulfation - glutathione conjugation - methylation - n-acteylation -

Answer 61

glucuronidation - 176 glycine - 57 sulfation - 81 glutathione conjugation -289 methylation -14 n-acteylation - 42

Question 62

What is the pka range which each of the following conjuagtions: glucuronidation glycine - sulfation - glutathione conjugation - methylation - n-acteylation -

Answer 62

glucuronidation - 3-3.5 glycine - 3.5-4 sulfation - less than 1 glutathione conjugation -2.1, 3.5 methylation - neutral n-acteylation - neutral

Question 63

In conjugation what happens to the lipohilicity?

Answer 63

usually decreases

Question 64

What can acidic conjugation lead to?

Answer 64

more albumin binding so decrease Vd

Question 65

What can increased polairty of the conjugate lead to?

Answer 65

limit partitioning into cells and this decreases the V

Question 66

What is the clearance of conjugated metabolites compared to parent drug?

Answer 66

tends to be higher and this si due to actyve excretion into urine or bile

Question 67

Would a metabolite be expected to have a half life longer or shorter than that of the parent drug?

Answer 67

drug(in blood) ---> metabolite (in blood) ----> metabolite in urine and bile t1/2 = ln2*V/CL Shorter Half-Life: If the metabolite is more readily eliminated or has a higher rate of metabolism, it could have a shorter half-life than the parent drug. This might occur if the metabolite is more polar and excreted faster via the kidneys or bile. Longer Half-Life: In some cases, a metabolite can have a longer half-life than the parent drug, especially if the metabolite is more lipid-soluble, is less readily excreted, or binds to tissue receptors and stays in the body longer. Some metabolites, especially active ones, can persist in the system for longer periods than the parent drug itself. -varies depends on drug

Question 68

What is N-acetyltransferase?

Answer 68

a soluble enzyme and isoniazid and sulfamethoxazole are substrates and genetic variation occurs have fast and slow acetylators -acetyl co enzyme a is the endogenous donor molecules -addition of acetyl group and acetyl coA which comes from glycolysis so do not eat sugar cannot make accoa and get rid of this

Question 69

Where does the acetyl group come from in NAT?

Answer 69

-acetyl co enzyme a is the endogenous donor molecules -addition of acetyl group and acetyl coA which comes from glycolysis so do not eat sugar cannot make accoa and get rid of this

Question 70

Can NAT conjugate AcCoA to an amino acid?

Answer 70

yes -if you add an amino acid into a fatty drug you can make it transported and this is why you take a fatty drug and add a glycine or valine and conjugate it aka exsicliver - acyclic nucleoside for antivirals and is used for herpes simplex put a valine on it and then the absorption increases a great deal

Question 71

What is the main metabolite of aspirin>

Answer 71

salicyluric acid the glycine conjugate of salicylic acid - 76% of aspirin is metabolized through amino acid conjugation

Question 72

What is salicylic acid the product of?

Answer 72

the hydrolytic product of acetyl saliciylic acid or aspirin and is further metabolized -salicyl uric acid is teh glyine conjugate

Question 73

What are some other metabolites of aspirin?

Answer 73

acyl glucuornide conjugate -phenol glucuornide conjugate -ring hydorylxated product -ring hydroyxlated product of glycine conjuagte shorter half life in blood if water soluble which is excreted in the urine

Question 74

What is the biosynthesis of glutathione?

Answer 74

formation of amino acids -glycine biosynthesis and cysteine biosynthesis and glutamate biosynthesis -the dipeptide formarition of glutamate with cystseine via gamma glutamyl synthesis gGS -glycine attachement to form glutathione -glutathione recycled from GSSG to GSH

Question 75

What is the drug interaction with glutathione?

Answer 75

GSH + Drug GS-Drug Drug-Cys-Gly (glutamic acid leaves) Drug-Cys (glycine leaves) Drug-NAC (acetyl n-cysteine)

Question 76

What are sulfotransferases?

Answer 76

co factor is paps - a high energy intermesiate low levels in vivo but made quickly from inorganic sulfate or catabolism of cysteine and methionine cosubstrate depletion - reduced inorganic sulfate or cysteine secondary to gsh depletion -add a sulfate to OH group

Question 77

What is transulfuration?

Answer 77

cyanide + thiosulfate -----> thiocyanate + sulfite -mediated by mitochondrial thiosulfate sulfurtransferase

Question 78

What is sulfation?

Answer 78

-major conjugation pathway for phenol and alcohols and amines compounds can be glucuronidated can also be sulfated can be competition between two paths -in general the sulfate conjugation predominates at low substrate concentration and glucuronide conjugate predominates at high substrate concentration -conducted by the soluble enzyme sulfotransferase -endogenous donor molecule to conjugation is paps -non inducible

Question 79

Where are sulfotransferases located and what are they induced by?

Answer 79

located - high levels in liver notable in intestine common throughout the body a cytosolic enzyme not membrane bound -induction - noninucible by Pb, BHA, 3MC, but PCN induction happens and is regulated by PXR

Question 80

What are substrates for sulfotransferases?

Answer 80

phenols and analine type amines are sulfated -phenols have both glucuronidation and sulfation as competing conjugation reactions -sulfation is often a high affinity low capcity pathay while glucuornidation is low affinity high capacity -low doses sulfation - high doses glucuronidation -sulfates are excreted in the urine through some bile aid sulfates are excreted into bile

Question 81

What are some properties of sulfate metabolites?

Answer 81

usually inactive but minoxidil sulfate is actuve sulfates are stribg acids pka<1 as an acid sulfates bind to albumin and plasma protein binding is higher than of parent compound -excreted by kidney and larger molecules in bile -sulfation can have reversible metabolism -sulfation of hydroxylated aromatic amines can lead to reactive intermediates and putative toxicity

Question 82

Sulfate conjugation of acetaminophen happens how?

Answer 82

p450 oxidation , gsh conjugation, cleavage

Question 83

Why do we sulfate a nucleotide?

Answer 83

cause nucleotide is toxic but nucleoside is okay - mono nucleotides are kidney toxic

Question 84

What can quaternary amines do?

Answer 84

Quaternary amine and heightens lysosomes and causes autophagy which is one of the key elements of cancer -sulfation can produce active metabolite through this

Question 85

In acetaminophen metabolism what does NAPQI do?

Answer 85

this binds to protein adults and cause oxidative stress

Question 86

What is the role of sulfation in steoridal hormones?

Answer 86

sulfation effects cholestrol metabolism -increased water solubility can alter distrbution in blood and tissues