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Measuring and Modeling Metabolism
Richard Kolanczyk & Greg Lien
The McKim Conference on the Use of QSARsand Aquatic Toxicology in Risk Assessment
June 27-29, 2006
McKim In Vitro / In Vivo Metabolism Work Plan
2
Research Plan
USEPANHEERL, MED-Duluth
FOR
IN VITRO AND IN VIVO XENOBIOTICBIOTRANSFORMATION IN FISH: IMPLICATIONS
FOR PHYSIOLOGICALLY BASED TOXICOKINETICMODELS
Project/Activity Name & Number: Sub-Objective-80101, Ecosystem Res.; Res. Area-8-062,Modeling-Effects
Project Leaders: J. McKim, P. Schmieder, J. NicholsTheme Title: Biochemical and Cellular Toxicity & Toxicokinetics and Dosimetry
Theme Leader: P. Schmieder, J. NicholsInstitution: NHEERL/MED-Duluth
MicrosomesCytosol
S-9
Isolated Cells &
Cell Lines
Isolated Perfused
Individual
Metabolism
Isolated Enzyme
Linkages Across Levels of Biological Organization
Molecular Cellular Organ Organism
Understanding
Relevance
Subcellular
Slices
Tissue
Across Various Endpoints
3
Extrapolation Issues
1.) Species
2.) Maturation
3.) Gender
4.) Scaling
5.) Chemical
Jim’s Question:
“Do species differences have significant effects on
the hepatic Phase I and II
biotransformation of xenobiotics by fish?”
4
MicrosomesCytosol
S-9
Isolated Cells &
Cell Lines
Isolated Perfused
Individual
Metabolism
Isolated Enzyme
Linkages Across Levels of Biological Organization
Molecular Cellular Organ Organism
Understanding
Relevance
Subcellular
Slices
Tissue
Across Various Endpoints
Three Species Comparison Study
“A comparative study of phase I and II hepatic microsomal biotransformation of phenol
in three species of salmonidae: hydroquinone, catechol, and phenylglucuronide
formation.”
5
Characterization of Microsomes
Isolated from Three Species of Fish
6.8 ±±±± 1.10.46 ±±±± 0.0310.8 ±±±± 0.8 *Lake Trout
5.6 ±±±± 1.10.51 ±±±± 0.049.1 ±±±± 0.5 *Brook Trout
10.9 ±±±± 5.00.38 ±±±± 0.0717.0 ±±±± 0.9 *Rainbow Trout
EROD
(pmol/min/mg)
P450 Protein
(nmol/mg)
Microsomal
Protein
(mg/g liver)
SPECIES
CHARACTERIZATION:
For each species N = 6 fish; all measurements done in triplicate.
* Microsomal Protein mg/g liver (P<0.0001) RBT different from BKT and LKT
OH
OGLUC
OH
OH
OH
OH
phenylglucuronide
phenol
catecholhydroquinone
[CAT]
[PG]
[HQ]
6
Michaelis-Menten Kinetics for Phase I Metabolism of Phenol in Three Species of Fish
653 ±±±± 10243 ±±±± 22 *Lake Trout
1099 ±±±± 23342 ±±±± 23 *Brook Trout
742 ±±±± 8916 ±±±± 7 *Rainbow Trout
Vmax (pmol/min/mg)Km (mM)SPECIES
HYDROQUINONE:
124 ±±±± 1920 ±±±± 12 *Lake Trout
155 ±±±± 3127 ±±±± 17 *Brook Trout
150 ±±±± 169 ±±±± 4 *Rainbow Trout
Vmax (pmol/min/mg)Km (mM)SPECIES
CATECHOL:
For each species N = 6 fish; all measurements done in triplicate.
Km and Vmax fitted to the average rate (over six preparations) at each phenol concentration.
* Km HQ & CAT (P<0.01) RBT different from BKT and LKT
Michaelis-Menten Kinetics for Phase II Metabolism of Phenol in Three Species of Fish
708 ±±±± 152 *7 ±±±± 7Lake Trout
1958 ±±±± 423 *8 ±±±± 7Brook Trout
1605 ±±±± 450 *11 ±±±± 10Rainbow Trout
Vmax (pmol/min/mg)Km (mM)SPECIES
PHENYLGLUCURONIDE:
For each species N = 6 fish; all measurements done in triplicate.
Km and Vmax fitted to the average rate (over six preparations) at each phenol
concentration.
* Vmax PG (P<0.01) LKT different from RBT and BKT
7
Summary of Species Comparison
• Rainbow Trout (RBT), Brook Trout (BKT) and Lake Trout (LKT) all metabolized phenol through the same pathways
• While the Km values for conjugation of phenol are similar across species, the lower Vmax in LKT means they will saturate their conjugation system at lower concentrations of phenol and may be more susceptible to the toxic effects of Phase I metabolites.
Jim’s Question:
“Do gender and sexual maturation have
significant effects on the
hepatic Phase I and II biotransformation of
xenobiotics?”
8
MicrosomesCytosol
S-9
Isolated Cells &
Cell Lines
Isolated Perfused
Individual
Metabolism
Isolated Enzyme
Linkages Across Levels of Biological Organization
Molecular Cellular Organ Organism
Understanding
Relevance
Subcellular
Slices
Tissue
Across Various Endpoints
Brook Trout Maturation Study
• 6 fish monthly - 3 males & 3 females• Fish held on Duluth photoperiod – June to December• Blood drawn • Body Weight, Gonad Weight, Liver Weight measured• Liver Microsomal Preparations
9
June July Aug Sept Oct Nov Dec0
10
20
30
40
50
60
70
ng
/ml p
lasm
a
HORMONE LEVELS - ESTRADIOL
June July Aug Sept Oct Nov Dec0
10
20
30
40
50
60
70
ng
/ml p
lasm
a
TESTOSTERONE
June July Aug Sept Oct Nov Dec0
10
20
30
40
50
60
70
80
ng/m
l pla
sm
a
11-KETOTESTOSTERONE
Females
Males
June July Aug Sept Oct Nov Dec0
1
2
3
4
Liv
er
Weig
ht/B
ody W
eig
ht (%
)
HEPATOSOMATIC INDEX [HSI]
June July Aug Sept Oct Nov Dec0
10
20
30
40
Gonad W
eig
ht/B
ody W
eig
ht (%
)
GONADOSOMATIC INDEX [GSI]
June July Aug Sept Oct Nov Dec0
5
10
15
20
mg/m
l pla
sm
a
VITELLOGENIN
Females
Males
10
OH
OGLUC
OH
OH
OH
OH
phenylglucuronide
phenol
catecholhydroquinone
[CAT]
[PG]
[HQ] June July Aug Sept Oct Nov Dec0
500
1000
1500
2000
2500
3000
pm
ol
HQ
/min
/mg
mic
ros
om
al
pro
tein
+/-
std
err
Vmax [HQ production]
males females
June July Aug Sept Oct Nov Dec0
200
400
600
800
1000
nm
ol
HQ
/min
/to
tal
liv
er
+/-
std
err
Vmax [HQ production]
males females
JUNE JULY AUG SEPT OCT NOV DEC0
50
100
150
200
250
300
Ph
en
ol (
mM
) +
/- s
td e
rr
Hydroquinone Production [Km]
MALES FEMALES
Females
Males
11
JUNE JULY AUG SEPT OCT NOV DEC0
20
40
60
80
100
Ph
en
ol (
mM
) +
/- s
td e
rr
Catechol Production [Km]
MALES FEMALES
June July Aug Sept Oct Nov Dec0
100
200
300
400
pm
ol C
AT
/min
/mg
mic
ros
om
al p
rote
in +
/- s
td e
rr Vmax [CAT production]
males females
June July Aug Sept Oct Nov Dec0
25
50
75
100
125
nm
ol
CA
T/m
in/t
ota
l li
ve
r +
/- s
td e
rr
Vmax [CAT production]
males females
Females
Males
JUNE JULY AUG SEPT OCT NOV DEC0
2
4
6
8
10
Ph
en
ol (
mM
) +
/- s
td e
rr
Phenylglucuronide Production [Km]
MALES FEMALES
June July Aug Sept Oct Nov Dec0
500
1000
1500
2000
2500
3000
pm
ol
PG
/min
/mg
mic
ros
om
al
pro
tein
+/-
std
err Vmax [PG production]
males females
June July Aug Sept Oct Nov Dec0
100
200
300
400
nm
ol
PG
/min
/to
tal
liv
er
+/-
std
err
Vmax [PG production]
males females
Females
Males
12
Summary of Maturation Study
• Through the maturation cycle Phase I metabolism increased while Phase II metabolism decreased in females. Therefore the female fish may be more susceptible to the toxic effects of Phase I metabolites.
Jim’s Question:
“Can we use fish liver slices to help us better
understand and predict
metabolism?”
13
MicrosomesCytosol
S-9
Isolated Cells &
Cell Lines
Isolated Perfused
Individual
Metabolism
Isolated Enzyme
Linkages Across Levels of Biological Organization
Molecular Cellular Organ Organism
Understanding
Relevance
Subcellular
Slices
Tissue
Across Various Endpoints
Liver
Liver Slices
Multi-well Tissue
Culture Plate
Metabolism in Liver Slices
14
OPP ChemicalsPredicted inactive
parent; “activated”metabolites
Predicted Metabolites
Existing Metabolism Simulator
Existing ER Binding Model
Prioritized Chemicals
Verified maps
Project Goal: Enhance Metabolic Simulator for EPA Regulatory Lists
Trout
liver slice
Rat
liver microsomes,S9
enhance
Expert Judgement
Analytical
methods
Verified ERactivation
Existin
g ER
Binding Model
simulator
improveER model
Cooperative Effort with LMC (O. Mekenyan) & NERL-Athens (J. Jones)
4,4'-diaminodiphenylmethane
“MDA”
Metabolism in Liver Slices
NH2
H2N
15
N H 2
H 2 N
N H 2
N H
C H 3
O
N H
H 3 C
O
N H
C H 3
O
N H 2
N H
O H N H 2
N
O HH 3 C
O
N H 2
NO
N H 2
O H
H 2 N
3-OH-MDA4,4'-diaminodiphenylmethane
“MDA”N-monoacetyl-MDA “MDA-MA” N, N-diacetyl-MDA
“MDA-DA”
N-hydroxy-N-acetyl-MDA
MDA-hydroxylamine
Proposed metabolic pathway for MDA
Nitroso-MDA
“MDA-NO”
N H 2
H 2 N
N H 2
N H
C H3
O
N H
H3
C
O
N H
C H3
O
N H 2
N HO H N H 2
N
O HH 3 C
O
N H 2
NO
N H 2
O H
H 2 N
3-OH-MDA4,4'-diaminodiphenylmethane
“MDA”N-monoacetyl-MDA “MDA-MA” N, N-diacetyl-MDA
“MDA-DA”
N-hydroxy-N-acetyl-MDA
MDA-hydroxylamine
Metabolites predicted to bind ER
Nitroso-MDA
“MDA-NO”
16
N H 2
H 2 N
N H 2
N H
C H3
O
N H
H3
C
O
N H
C H3
O
N H 2
N HO H N H 2
N
O HH 3 C
O
N H 2
NO
N H 2
O H
H 2 N
3-OH-MDA4,4'-diaminodiphenylmethane
“MDA”N-monoacetyl-MDA “MDA-MA” N, N-diacetyl-MDA
“MDA-DA”
N-hydroxy-N-acetyl-MDA
MDA-hydroxylamine
Measured MDA Metabolism
within the Liver Slice
Nitroso-MDA
“MDA-NO”
4,4'-diaminodiphenylmethane
“MDA”
N H 2
H 2 N
N H 2
N H
C H3
O
N H
H3
C
O
N H
C H3
O
N H 2
N
O HH 3 C
O
N-monoacetyl-MDA
“MDA-MA”N, N-diacetyl-MDA
“MDA-DA”
N-hydroxy-N-acetyl-MDA
Proposed metabolic pathway for MDA leading to VTG induction
17
4,4'-diaminodiphenylmethane
“MDA”
N H 2
H 2 N
N H 2
N H
C H3
O
N H
H3
C
O
N H
C H3
O
N H 2
N
O HH 3 C
O
N-monoacetyl-MDA
“MDA-MA”N, N-diacetyl-MDA
“MDA-DA”
N-hydroxy-N-acetyl-MDA
Proposed metabolic pathway for MDA leading to VTG induction
MDA
-10 -9 -8 -7 -6 -5 -4 -3 -2
-20
0
20
40
60
80
100
120
E2-50
MDA-1
E2-52
MDA-2
E2-63
MDA-3
Log Concentration (M)
[3H
]-E
2
Bin
din
g (
%)
MDA does not bind to rbt-ER
in Competitive Binding Assay
4,4'-diaminodiphenylmethane
“MDA”
N H 2
H 2 N
N H 2
N H
C H3
O
N H
H3
C
O
N H
C H3
O
N H 2
N
O HH 3 C
O
N-monoacetyl-MDA
“MDA-MA”N, N-diacetyl-MDA
“MDA-DA”
N-hydroxy-N-acetyl-MDA
Proposed metabolic pathway for MDA leading to VTG induction
MDA
-10 -9 -8 -7 -6 -5 -4 -3 -2
-20
0
20
40
60
80
100
120
E2-50
MDA-1
E2-52
MDA-2
E2-63
MDA-3
Log Concentration (M)
[3H
]-E
2
Bin
din
g (
%)
MDA does not bind to rbt-ER
in Competitive Binding Assay
CT
RL
1.0×103
1.0×104
1.0×105
1.0×106
1.0×107
1.0×108
MDA
-10 -9 -8 -7 -6 -5 -4 -3 -2
MDA
E2
crtl
MDA
E2
ctrl
MDA
E2
crtl
Log Concentration (M)
Vtg
mR
NA
(co
py
#/4
00
ng
to
tal
RN
A)
Trout liver slices exposed to MDA produce Vtg
18
4,4'-diaminodiphenylmethane
“MDA”
N H 2
H 2 N
N H 2
N H
C H3
O
N H
H3
C
O
N H
C H3
O
N H 2
N
O HH 3 C
O
N-monoacetyl-MDA
“MDA-MA”N, N-diacetyl-MDA
“MDA-DA”
N-hydroxy-N-acetyl-MDA
Proposed metabolic pathway for MDA leading to VTG induction
MDA-DA
-11 -10 -9 -8 -7 -6 -5 -4 -3 -20
10
20
30
40
50
60
70
80
90
100
110
120
E2-63MDA-DA-1
Log Concentration (M)
[3H
]-E
2
Bin
din
g (
%)
MDA-DA does not bind to rbt-ER in competitive binding assay
4,4'-diaminodiphenylmethane
“MDA”
N H 2
H 2 N
N H 2
N H
C H3
O
N H
H3
C
O
N H
C H3
O
N H 2
N
O HH 3 C
O
N-monoacetyl-MDA
“MDA-MA”N, N-diacetyl-MDA
“MDA-DA”
N-hydroxy-N-acetyl-MDA
Proposed metabolic pathway for MDA leading to VTG induction
MDA-DA
-11 -10 -9 -8 -7 -6 -5 -4 -3 -20
10
20
30
40
50
60
70
80
90
100
110
120
E2-63MDA-DA-1
Log Concentration (M)
[3H
]-E
2
Bin
din
g (
%)
MDA-DA does not bind to rbt-ER in competitive binding assay
CT
RL
1.0×10 3
1.0×10 4
1.0×10 5
1.0×10 6
1.0×10 7
1.0×10 8
MDA-DA metabolite
-10 -9 -8 -7 -6 -5 -4 -3 -2
MDA-DA
E2
ctrl
MDA-DA solubilty limit
Log Concentration (M)
Vtg
mR
NA
(co
py
#/4
00
ng
to
tal
RN
A)
MDA-DA does not produce VTG
19
4,4'-diaminodiphenylmethane
“MDA”
N H 2
H 2 N
N H 2
N H
C H3
O
N H
H3
C
O
N H
C H3
O
N H 2
N
O HH 3 C
O
N-monoacetyl-MDA
“MDA-MA”N, N-diacetyl-MDA
“MDA-DA”
N-hydroxy-N-acetyl-MDA
Proposed metabolic pathway for MDA leading to VTG induction
MDA-MA
-11 -10 -9 -8 -7 -6 -5 -4 -3 -20
20
40
60
80
100
120
140
160
E2-67MDA-MA-1
E2-69
MDA-MA-2
Log Concentration (M)
[3H
]-E
2
Bin
din
g (
%)
MDA-MA does not bind to rbt-
ER in competitive binding assay
4,4'-diaminodiphenylmethane
“MDA”
N H 2
H 2 N
N H 2
N H
C H3
O
N H
H3
C
O
N H
C H3
O
N H 2
N
O HH 3 C
O
N-monoacetyl-MDA
“MDA-MA”N, N-diacetyl-MDA
“MDA-DA”
N-hydroxy-N-acetyl-MDA
Proposed metabolic pathway for MDA leading to VTG induction
MDA-MA
-11 -10 -9 -8 -7 -6 -5 -4 -3 -20
20
40
60
80
100
120
140
160
E2-67MDA-MA-1
E2-69
MDA-MA-2
Log Concentration (M)
[3H
]-E
2
Bin
din
g (
%)
MDA-MA does not bind to rbt-
ER in competitive binding assay
CT
RL
1.0×10 3
1.0×10 4
1.0×10 5
1.0×10 6
1.0×10 7
1.0×10 8
MDA-MA metabolite
-10 -9 -8 -7 -6 -5 -4 -3 -2
MDA-MA
E2
crtl
Log Concentration (M)
Vtg
mR
NA
(co
py
#/4
00
ng
to
tal
RN
A)
Vtg induction upon
exposure to MDA-MA
20
• Both MDA and mono-acetylated MDA are further bioactivated in the liver slice.
• Proposed metabolic route is N-acetylation of
MDA followed by N-hydroxylation.
MDA Summary
N H 2
N
O HH 3 C
O
N-hydroxy-N-acetyl-MDA
Acknowledgements
U.S. EPA
Jim McKim
Pat Schmieder
Mark Tapper
Alex Hoffman
Doug Kuehl
Barb Sheedy
Jeff Denny
NRC Post Docs
Laura Solem
Hristo Aladjov
Student Services Contractors
Beth NelsonVictoria Wehinger
Luke Toonen
Ben Johnson
21
MicrosomesCytosol
S-9
Isolated Cells &
Cell Lines
Isolated Perfused
Individual
Metabolism
Isolated Enzyme
Linkages Across Levels of Biological Organization
Molecular Cellular Organ Organism
Understanding
Relevance
Subcellular
Slices
Tissue
Across Various Endpoints
Acknowledgements
• James McKim Sr.
• James McKim Jr.
• John Nichols
• Alex Hoffman
• Doug Kuehl
• Laura Solem
• Richard Kolanczyk
• Correne Jenson
22
Jim’s Question:
“ Can an isolated perfused liver
can serve as a representative model for studies of xenobiotichepatic biotransformation in fish? ”
Isolated Perfused Liver
• an intact organ
• bile flow and concentration
• can control perfusion flow and constituents
• single pass
• easily scaled to whole fish
23
Isolated Perfused Liver
• can’t use whole blood
• must supply oxygen
• special apparatus
• viability assays
• short time frame
0.5% CO2 / 99.5% O2
Flow Transducer
Pressure Transducer
Liver
Pump
Gas
Out
24
Summary of Isolated Perfused Rainbow Trout Liver Experiments
55Number of experiments
4.482 ± 0.5264.432 ± 0.299Liver Weight (mean±SE, g)
2-male, 3-female3-male, 2-femaleFish Gender
667 ± 51691 ± 62Fish Weight (mean±SE, g)
NonpulsatilePulsatileParameter
Profile of perfusion medium afferent to the liver (mean±SE) for pulsatile and nonpulsatile isolated rainbow trout liver experiments
7.784 ± 0.0027.787 ± 0.006pH (n=50)
1.40 ± 0.0031.46 ± 0.004Perfusion Flow Rate (mL/min/g-liver) (n=3000)
10.95 ± 0.00210.92 ± 0.001Temperature (°C) (n=3000)
134 ± 2134 ± 27-Ethoxycoumarin (µmol/L) (n=50)
10.2 ± 0.0210.3 ± 0.04Glucose (mmol/L) (n=50)
304.2 ± 1.0306.2 ±0.9Osm (mmol/kg) (n=50)
125 ± 0.4124 ± 0.5Cl- (mmol/L) (n=50)
0.94 ± 0.010.95 ± 0.01Ca2+ (mmol/L) (n=50)
147.0 ± 0.5147.9 ± 0.5Na+ (mmol/L) (n=50)
5.30 ± 0.025.34 ± 0.02K+ (mmol/L) (n=50)
3.7 ± 0.033.6 ± 0.05pCO2 (mmHg) (n=50)
473.3 ± 3.9473.5 ± 3.8pO2 (mmHg) (n=50)
NonpulsatilePulsatileParameter
25
1 2 3 4 5 6 7 8 9 10
h
0
1
2
3
cm
H2O
/mL/m
in
Resistance for isolated perfused livers (n=5)
Non-pulsatile
* * ** * * *
Pulsatile
1 2 3 4 5 6 7 8 9 10
h
0
10
20
30
40
um
ol O
2/h
/g w
et
wt
liver
Oxygen consumption of isolated perfused livers (n=5)
*
Non-pulsatile Pulsatile
26
1 2 3 4 5 6 7 8 9 10
h
-30
-20
-10
0
10
20
30
40
50
60
µm
ol/h/g
-liv
er
Glucose flux from isolated perfused livers (n=5)
Non-pulsatile
*
Pulsatile
1 2 3 4 5 6 7 8 9 10
h
-3
-2
-1
0
1
2
3
4
5
6
7
8
µm
ol/h/g
-liv
er
Potassium flux from isolated perfused livers (n=5)
**
Non-pulsatile Pulsatile
27
1hr 2hr 3hr 4hr 5hr 6hr 7hr 8hr 9hr 10hr0
10
20
30
40
(nm
ol/g/h
)
Efflux of 7-Hydroxycoumarin from Isolated Perfused Livers and Slices
Non-pulsatile Pulsatile Slices
1hr 2hr 3hr 4hr 5hr 6hr 7hr 8hr 9hr 10hr0
100
200
300
400
(nm
ol/g/h
)
Efflux of 7-Hydroxycoumarin Glucuronide from Isolated Perfused Liver and Slices
Non-pulsatile Pulsatile Slices
28
Summary of Isolated PerfusedLiver System
• The function and viability of the system were preserved for the entire 10h period
• Pulsatile perfusion did not significantly improve the function or viability of the system
• An isolated perfused liver can serve as a representative model for studies of xenobiotichepatic biotransformation in fish
MicrosomesCytosol
S-9
Isolated Cells &
Cell Lines
Isolated Perfused
Individual
Metabolism
Isolated Enzyme
Linkages Across Levels of Biological Organization
Molecular Cellular Organ Organism
Understanding
Relevance
Subcellular
Slices
Tissue
Across Various Endpoints
29
Jim’s Question:
“ Can microdialysistechniques be used to
estimate
biotransformation rate and capacity in fish ? ”
Use of Microdialysis Methods to Estimate Biotransformation Rate
and Capacity in Fish.
In Vivo Rate of Phenol Glucuronidation by Rainbow Trout
30
4 24 48
Time (h)
0
100
200
300
400
µm
ol/
L
Concentration of Phenol and Metabolites in Blood of Rainbow Trout
Phenol Phenylglucuronide Phenylsulfate
MICRODIALYSIS PROBE IN THE DORSAL AORTA
OF A RAINBOW TROUT.
(roof of mouth)
Probe Guide
and Probe
Microdialysis
Inlet Tubing
Suture
Suture
MicrodialysisOutlet Tubing
Dorsal Aorta Gill Arch 4Gill Arch 3Gill Arch 2Gill Arch 1
4mm Microdialysis Membrane
Microdialysis
Outlet Tubing
Suture
Suture
Microdialysis
Inlet Tubing
Suture
ENLARGEMENT OF
PROBE GUIDE AND PROBE
Microdialysis Probe
Probe Guide
31
PG concentration time-course in plasma (") and urine (!).
0 10 20 30 40 50 60 70
Time (h)
0
50
100
150
200
PG
Co
ncen
trati
on
(u
g/m
l)
32
Elimination mass-balance for infused phenylglucuronide (PG)
Fish PG infused(µg) a
PG in urine(µg) b
PG in bile(µg) c
urine + bilePG infused
1 19,802 17,592 unavailable 0.89
2 19,934 19,732 174 1.00
3 21,352 17,747 263 0.84
4 18,018 17,807 252 1.00a Stock concentration (µg/mL) x flow rate (mL/h) x 24 hb Summed mass of PG in all urine samplesc Concentration in bile at test termination (µg/mL) x bile volume (mL)
0 10 20 30 40 50 60 70
Time (h)
0.00
0.50
1.00
1.50
2.00
Lo
g10 P
G C
on
c.
(ug
/ml)
33
PROGRAM Biotransformation ModelINITIAL
!Volume of central compartmentCONSTANT VC1 = 0.25 !Liters!Formation RateCONSTANT Kf = 2.059 !umol/h!Elimination rate constantCONSTANT K10 = 0.054 !/t
END ! of initialDYNAMIC
ALGORITHM IALG = 2NSTEPS NSTP = 1000CINTERVAL CINT = .001
DERIVATIVE!Rate of change in central compartmentRCC1 = Kf - (AC1*k10) !umol/h!Amount of chemical in central compartmentAC1 = integ(RCC1, 0.0) !umol!Concentration of chemical in central compartmentCC1 = AC1/VC1 !umol/L
END ! of derivative CONSTANT TSTOP = 48 TERMT( T .GE. TSTOP )END ! of dynamicEND ! of program
34
Results of parameter estimation for biotransformation model
Fish ID k1010aa VDbb kff
cc
1dd .098 .1952.390
2dd .063 .1954.535
3dd .046 .1953.006
4ee .046 .1952.621
5ee .046 .1953.339
6ee .046 .1952.837
7ee .046 .1953.565
aElimination rate constant (/h/kg). Lower bound, starting value, and upper bound values were0.046, 0.068, and 0.098, respectively
bVolume of distribution (L/kg). Lower bound, starting value, and upper bound values were 0.195,0.222, and 0.283, respectively
cBiotransformation rate (�mol/h/kg). Lower bound, starting value, and upper bound values were0.0, 2.0, and Inf., respectively
dIndwelling microdialysis probeePlasma samples
Results
• PG is the major Phase II metabolite of PH in rainbow trout.
• PG is eliminated from fish primarily in the urine
• The apparent rate of PG formation in rainbow trout ranges from 2.4 to 4.5 umol/h/kg at a PH exposure concentration of 50 umol/L. This corresponds to a first-order rate constant of approximately 0.08/h/kg.
• The mean rate of PG formation, when expressed in terms of hepatic microsomal protein, (assuming 14g liver/kg fish and 17 mg protein/g liver) is 280 pmol/min/mg protein). The rate derived from in vitro rainbow troutmicrosomal glucuronidation of PH and extrapolated to the same PH concentration as used in this study (50 umol/L) is 268 pmol/min/mg protein (R. Kolanczyk - personal communication).
35
Conclusions
• Microdialysis is an excellent technique for obtaining in vivo metabolism data. This technique allows for continuous sampling of unbound chemicals, the exclusion of most proteins (simplifying quantitative analysis), high temporal resolution, and no net loss of fluid from the animal.
• In vivo rates of PH glucuronidation in rainbow trout were derived from plasma PG concentrations in fish dosed with PH, combined with knowledge of PG elimination rate and apparent volume of distribution. This kinetic technique provides a relatively simple method for estimating in vivobiotransformation rates in fish.
Jim’s Question:
“ Can a microdialysisprobe, implanted in the
liver, be used to estimate
hepatic biotransformation rate and capacity in fish ?
”
36
Use of Microdialysis Methods to
Estimate Biotransformation Rate
and Capacity in Fish.
Development of a Mathematical Model for Quantitative
Microdialysis
Inlet
OutletShaft
Laser-drilledHole
Hepatocytes
LiverTissue
PH
CA
PH
HQ
Illustration of the delivery of PH to the intact liver and the simultaneous
recovery of HQ and CA from the hepatocytes into the dialysate.
37
min.
nmol/mL
Concentration of Phenol delivered to the probe vs. time
0000 50505050 100100100100 150150150150 200200200200
PH in Perfusate (mM)
0000
1111
2222
3333
4444
5555
6666
7777
8888
HQ in dialysate
(pmol/min)
Liver Microdialysis
38
Objective
• develop a mathematical model to estimate
concentration profiles of parent and metabolites
around a MD probe implanted in liver
• relate an absolute recovery of metabolites in dialysate (pmol/min) with a biotransformation
rate in a specific metric of tissue (ml or g)
CCCCddddinininin
CCCCddddoutoutoutout
BloodBloodBloodBlood
VesselVesselVesselVessel
Uptake and MetabolismUptake and MetabolismUptake and MetabolismUptake and Metabolism
DiffusionDiffusionDiffusionDiffusion
HepatocyteHepatocyteHepatocyteHepatocyte
39
Parameters for Quantitative Microdialysis Model
• physical dimensions
• perfusate flow rate
• diffusivity
• partitioning
• blood flow
• biotransformation
• microvascular exchange properties
40
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0.36 - 0.4
0.32 - 0.36
0.28 - 0.32
0.24 - 0.28
0.2 - 0.24
0.16 - 0.2
0.12 - 0.16
0.08 - 0.12
0.04 - 0.08
0 - 0.04
mm
mm
nmol/mL
Contour graph of CAT concentration around a MD probe in liver
41
0 50 100 150 200
PH (mM)
0
20
40
60
80
PH
(m
M)
fish 5
fish 8
fish 9 fish 10 Predicted
Measured and predicted PH in dialysate
0 50 100 150 200
PH (mM)
0
1
2
3
4
5
6
7
8
HQ
(pm
ol/m
in)
fish 5
fish 8
fish 9 fish 10 Predicted
2.76.0
Measured and predicted absolute recovery
Km(mM) =Vmax(nmol/min/ml liver) =
42
0 50 100 150 200
PH (mM)
0
0.1
0.2
0.3
0.4
0.5
0.6
Ca
t (p
mo
l/min
)
fish 5
fish 8
fish 9 fish 10 Predicted
5.30.4
Measured and predicted absolute recovery
Km(mM) =Vmax(nmol/min/ml liver) =
43
Summary
• Microdialysis is a very general technique for sampling and delivering small molecules in almost any tissue or body fluid.
• Microdialysis provides a "window" into conditions at the tissue level and "purifies" the sample for simplified analysis.
• Microdialysis has a wide range of applications from basic research to clinical applications.
Summary
• With a finite difference MD mass transport model, delivery MD may be used to estimate in
vivo biotransformation rates.
• The modeling construct will accommodate non-
linear metabolism and provide for a continuous simulation through a series of concentrations
typical of Michaelis-Menten kinetics.
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