Importance Of Obtaining Information Prior To Determining Vmax, Km Values for Use In PBPK Models

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The Importance Of Obtaining Information Prior To
Determining Vmax Km Values for Use In PBPK Models

» Specific Content Of Tissue Enzymes Metabolizing Organophosphorus Pesticides A

Emerging Technologies

BSTRACT

James B. Knctak1, Rogelio Tornero-Velez2, Fred Power2, Jerry N. Blancato2 and Curtis C. Dary2,

'State University of New York at Buffalo, Buffalo, NY. 2U.S. EPA, Human Exposure and Atmospheric Sciences Division, Las Vegas, NV.

PBPK/PD models require metabolism

parameter values to represent certain tissue enzymes. Current litera-
ture values cannot be easily used in PBPK models because of differ-
ences in standard methodologies used in their determination. In prac-
tically all cases, the investigators failed to determine the specific
enzyme content in tissues of interest. For example, the concentration
of certain enzymes in plasma (PONi paraoxonase) may vary as much as
10 fold (60 to 600 jxg/ml) making it difficult to select proper enzyme-
substrate concentrations for the determination of kinetic parameters
(Vmax, Km). Recently, individual enzymes (CYPs 1A2, 2B6, 2D6 and
3A4) have been measured by spectral or immunochemical methods to
obtain Vmax, Kjq values for human whole liver microsomes.
Mathematical equations have been developed for expressing individual
CYP specific content and activity in native liver microsomes for use in
PBPK models.

ACKGROUND/OBJECTIVES

• Cytochrome p-450 isozymes (CYPs) located in liver microsomes catalyze
the activation of Organophosphorous Insecticides (OP) to toxic,
cholinesterase (ChE) inhibiting agents.

• A-esterases oxonases(PONi) located in brain, plasma and liver microsomes
catalyze the hydrolysis of toxic agents to nontoxic hydrolysis products.

• The nature (CYP form) and specific content (concentration/unit of tissue) of
these enzymes vaiy across species and within species making it difficult to

obtain reproducible and representative metabolic rate constants.

~ Advances in molecular biology provide a way to measure
the specific content and activity of these enzymes in

The objective of this poster is to present a sound
approach to obtaining data required in
PBPK/PD models (Figure 1).

¦m

HEHl

, Km) of reconstituted human CYPs (1A2, 2B6,

LIVER: P-450 ISOZYMES:

1. Determine the specific activity (Vm.

2C9*i, 2C19 and 3A4)

2. Use Hanes-Woolf plot ([S]/v versus [S]) or Eadie-Hofstee plot (v versus v/[S]) to
obtain Vmax, Km (Segel, 1993).

3. Express Vmax in pmol of metabolite per CYP/min and Km in M.

4. Determine specific content of CYPs (iA2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and
3A5) in human liver microsomes. Report content in pmol of CYP/mg of microsomal
protein (see Table 1).

5. Normalize data for each CYP using the following equation (specific activity of CYP *
specific content in microsomes) to give pmol of oxon, PNP, or TCP formed /min/mg
of microsomal protein.

NR-

pmolCYP
\_ mg * protein J

pmolCYP

If multiple number of CYPs are involved calculate Total NR as indicated below:
TNR = X(pmol oxon/min/pmol CYP * pmol CYP/mg microsomal protein) + (...) + (...)

PLASMA or SERUM: a-esterasE:

6. Obtain plasma or serum from blood donors.

7. Determine the specific content of A-esterases in blood plasma using methods of
Blatter-Garin et al., (1994), Costa et al., (1999) and Kujiraoka et al., (2000) involving
the separation (ELISA) and immunoquantitation of PONi.

8. Determine the specific activity of A-esterases (PONi). Report activity in pmol/hr/kg
of body weight.

9. Report activity in pmol/hr/kg of body weight for use in PBPK/PD models. Human
liver contains an average of 32 mg of microsomal protein/g of liver (Lipscomb and
Teuschler, 2000), while rat liver contains 47.5 mg of microsomal protein/g of liver
(Gentest Corp).

ISCUSSION AND
CONCLUSIONS

1. The specific content of individual CYPs, A-esterases, and PONi in tis-
sues varies considerably between individuals and is believed to be the
main reason for the poor reproducibility of Vmax, Km values in and
between laboratories. See Table 1.

2. The specific activity of hydrolytic enzymes in plasma (PONi, A-
esterase) is well known. However, recent studies have indicated the spe-
cific content of these enzymes in plasma varies between certain individ-
uals and needs to be determined.

3. The specific activity and content of A-esterases in liver microsomes need
to be determined according to the methods used for the plasma
enzymes.

EFERENCES

Blatter Garin M-C, Abbott C, Messmer S, Mackness M, Durrington P, Pometta D, James RW (1994)
Quantification of human serum paraoxonases by enzyme-linked immunoassay: population differences
in protein concentrations. Biochem J 304:549-554.

Costa LG, Li WF, Richter RJ, Shih DM, Lusis A, Furlong CE (i999) The role of paraoxonase (PONi) in
the detoxication of organophosphates and its human polymorphism. Chem-Biol Interaction 38:48.

Kujiraoka, T, Oka T, Ishihara M, Egashira T, Fujioka T, Saito E, Saito S, Miller NE, Hattori H (2000) A
sandwich enzyme-linked immunosorbent assay for human serum paraoxonase concentration. J Lipid
Res 41:1358-1363.

Lipscomb JC, Teuschler LK (2000) Integration of in vitro data on xenobiotic metabolism and tissue
enzyme recovery with physiologically based pharmacokinetic (PBPK) modeling to estimate human
interindividual pharmacokinetic variance. Annual Meeting of the Society of Risk Analysis. Washington,
DC, December 2000.

Segel IH (1993) Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State
Enzyme Systems. John Wiley & Sons, Inc., NY.

IGURE & TABLE

FIGURE 1. PBPK MODEL FOR PARATHION

TABLE 1. Specific Content of CYPs in Native Human Liver Microsomes (HLM)

CYP

HLM-3

HLM-23

HLM-24

HLM-34

HLM-43

HLM-56

AVE

1A2*

46.2

2A6

52.2

2B6*

15.6

2C9

50.5

2C19

17.0

2D6

14.0

2E1

51.6

3A4*

306

116

3A5

0.9

0.5

0.72

1.00

1.1

0.7

0.82

Data taken from Gentest Corp., Woburn, MA.

Specific Content in pmol of enzyme per mg of protein

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