50904989 MT5003

Drug metabolism in vitro

Aim:

To analyze in vitro metabolism of

Aminopyrine.

Introduction:

Aminopyrine is a heterocyclic pyrazolone compound and is

commonly used to assess the cytochrome P450 metabolic
activity as a liver function test. It measures the
detoxification function of the liver.

The main drug metabolizing enzymes are found in endoplasmic reticulum of

hepatocytes known as Cytochrome P450. Drugs are metabolized by the CYP
enzymes to make them more water soluble (Pubchem compounds,2009) to

facilitate their elimination, this is biotransformation (Pharmatutor, 2009).

Lipohphilic drugs are easily absorbed in gastrointestinal tract but cause

problems in elimination as they are reabsorbed in renal tubules. Main organs
involved in elimination are kidney (major part) and liver (minor part)
(Nottingham university,2005).

Drug metabolism could be divided into two phases-

Phase I metabolism where oxidation, reduction and hydrolysis reaction make

the drug molecule more polar to facilitate their rapid elimination. Most of
Phase I metabolism is carried out by Cyt P450 (Rang et. al.,2003).

Phase II metabolism are conjugation reaction also known as synthetic

reaction as it involves addition of small group to make the molecule water
soluble.

Cofactors are helper molecules which bind to the enzyme tightly and they
help in transformation. NADPH is used as cofactor for CYP P450 which
metabolizes Aminopyrine.

This experiment was done in mice where CYP2B1 metabolized

the substrate i.e Aminopyrine using Phenobarbital as an inducer
of the Cyp enzymes. NADPH, O2 and Mg2+ were added as co-
factor mixture (Goldstein et. al, 1974).

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Phenobarbital is a heterocyclic pyrimidine compound which is a barituric acid

derivative. It is weakly acidic and has a pKa of 7.4 (Rowland and
Towzer,1995). Phenobarbital has a low hepatic extraction ratio of <0.3
(Rowland and towzer,1995). It acts as a nervous system depressant, so
(Pubchem compounds,2009)

used as anticonvulsant, sedative and as GABA modulator. Data from animal

studies show that it is carcinogenic in humans (chemical carcinogenesis
research information centre,2009).

Phenobarbital increases the metabolism of other drugs by increasing the

synthesis of the drug metabolizing (Cytochrome) enzymes (Rowland and
towzer,1995).

Phenobarbital activates Aryl hydrocarbon receptors (AHR) these are cytosolic

transcription factors. When Phenobarbital binds to AhR (Roberts et.al., 1961)
X associated protein 2 is released and nuclear localization sequence is
exposed and then the ligand and AhR complex is translocated into the
nucleus (Denison et. al., 2002). Once inside the nucleus Hsp90 dissociates
this allows the complex to accommodate Ahr nuclear translocator (Denison
et. al., 2002). This activated complex can then interacts with DNA by binding
to specific sequences on the DNA known as Xenobiotic response elements
(XRE) in the promoter region and causes change in the gene expression
(Denison et. al., 2002).

Aminopyrine is converted into formaldehyde and 4-monomethyl pyridine in

phase II metabolism by N-demethylation (Goldstein et. al, 1974).

Enzyme kinetics plays a very important role in interpreting the results, some
of the important parameters are Vmax, Km.

Vmax is the maximum velocity of the reaction at which all enzymes get
saturated by substrate. Vmax give information about how fast the enzyme
can convert substrate to product when it is completely saturated with
substrate (Robert et. al., 2003)

Km is Michaelis Menten constant gives information about the affinity of the

substrate towards the enzyme. Km is inversely proportional to the affinity.
Km could be defined as the substrate concentration at which reaction has
acquired half of the maximum velocity. High km requires high concentration
of substrate to achieve Vmax and hence low affinity (Robert et. al., 2003).

Michaelis Meneten Equation

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Vo = Vmax[S]Km+[S] where Vmax is maximum rate of reaction

Vo is initial reaction rate

Km is Michaelis Menten constant

[S] is substrate concentration

Vmax and Km are important terms when considering a competitive inhibition

reaction and they help us in judging the fate of the substrate and whether
rate will be affected by availability of substrate.

Reaction showing the cyp mediated metabolism of Aminopyrine to

formaldehyde and 4-monomethly pyridine. Formaldehyde is an important
production which gives a measure of the enzyme function and is read
spectrophotometrically.

Figure2. Showing the metabolism of Aminopyrine by Cytochrome P450 2B1 resulting in the
formation of formaldehyde as the product. (pubchem compounds,2009)

Objective:

To study the effect of Phenobarbital on Cytochrome P450 function.

To study the effects of Phenobarbital on the metabolism of Aminopyrine.

Materials Required:

Formaldehyde solution (37 % (w/v))

Cofactor mixture (0.25mM NADP+ , 2.5mM DL- isocitric acid, 0.6

units isocitrate dehydrogenase, 5mM MgSO4,
0.1M phosphate buffer pH 7.4)

Aminopyrine 5mM & 40mM

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Trichloroacetic acid 20% (w/v)

Nash reagent 45g ammonium acetate, 0.6ml acetylacetone

and 0.9ml glacial acetic acid dissolved in water
to a final volume of 100ml.

Protocol:

Pre practical preparation to obtain Microsome

Male Sprague dawley rats were dosed with 80mg/Kg sodium

Phenobarbital wit saline for 3 days. On fourth days rats were killed to
isolate the liver and microsomes were prepared.

Microsome preparation

Buffers were pre chilled to 4 C and the isolation procedure was carried
out on ice. The liver were excised into washing buffer then
homogenized using homogenizing buffer using motor driven pestle and
tube.

The homogenates were then centrifuged at 9000g for 20 min at 4°C.

The supernatant was then centrifuged at 180,000g for 60 minutes at
4°C.

The supernatant obtained was discarded and the pellet was re-
suspended in the homogenizing buffer and centrifuged at 180,000g for
further 60 minutes at 4°C. The pellet was re-suspended in storage
buffer and stored at -80°C.

Measurement of total cytochrome P450

Microsomes were diluted and 1 ml was pipette into two cuvettes.

Baseline spectrum was obtained between wavelengths 390-500nm
using scanning spectrophotometer which is capable of measuring
turbid solutions.

Carbon monoxide was gently bubbled through the sample only for 30
seconds and few grains of sodium dithionite were added to both
sample and reference cuvettes. After a gentle mixing different
spectrum was obtained with an absorbance peak at 450nm.

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Absorbance was measured at 450nm and multiplied with dilution

factor.

Protocol for the experiment

1. 8 sample tubes were marked from 1-8 and solutions were added in
following order as shown in the table below.

Sample tube 1 2 3 4 5 6 7 8
number

Vol. water (μl) 75 50 0 75 50 0 0 100

Vol of buffer (ml) 0 0 0 0 0 0 1.85 1.8

5

Vol. of 50 50 50 50 50 50 50 50
microsomes (μl)

Vol of cofactor 1.85 1.85 1.85 1.85 1.85 1.85 0 0

mixture (ml)

2. All tubes were then vortex mixed and placed in shaking water bath at
37 C. Each tube was placed in water bath after an interval of 30
seconds.
3. After the initial pre-incubation of 4 minutes the reaction was initiated
by the addition of Aminopyrine in the amounts mentioned in the table
below.

1 2 3 4 5 6 7 8

Vol of 5mM 25 50 100 0 0 0 0 0

Aminopyrine (μl)

Vol of 40mM 0 0 0 25 50 100 100 0

Aminopyrine (μl)

4. All tubes were vortex mixed and were placed in water bath again.

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5. At the end of the 30 min incubation period of each tube, the reaction
was terminated by the addition of 0.5ml 20% TCA and sample tubes
were vortex mixed.
6. Once the reaction has been terminated, sample was centrifuged at
4000 rpm for 5 minutes to precipitate the microsomal protein.

Preparation of standard formaldehyde curve

7. 6 different sets of tubes were marked and formaldehyde and buffer
were added in following amounts mentioned in the table below. Given
formaldehyde solution was already diluted from 37% (w/v) to 0.0037%.

Standard No. S1 S2 S3 S4 S5 S6

Vol of standard 0 25 50 75 100 125

formaldehyde (μl)

Vol of buffer (ml) 2.0 1.975 1.95 1.925 1.9 1.875

nmol of 0 31 62 92 123 154

HCHO/2ml

8. 0.5 ml of 20% TCA was added to all the tubes and vortex mixed.

Nash Reaction
9. 8 set of fresh tubes were labeled for sample and 6 set of fresh tubes
for standard.
10. 1.5ml Nash reagent was added to all the tubes.
11. 1.5ml of sample and standard supernatant was pipette to the
corresponding tubes containing Nash regent. All tubes are vortex
mixed.
12. Standard and sample tubes were incubated for 10 min in hot water
bath at 60 C and then allow it cool at room temperature.
13. Absorbances of the solutions are measured spectrophotometrically at
412nm using distilled water as reference (refer table 1 & graph 1).

Determination of Protein by Lowry Method

Materials Required

1. Sodium hydroxide (NaOH) 0.3 M

2. BSA (bovine serum albumin) 250 g/ ml in 0.3M NaOH

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3. Copper Sulphate (CuSO4) 1% (w/v)

4. Sodium potassium tartrate 2% (w/v)
5. Sodium carbonate (Na2CO3) 2% (w/v)
6. Reagent A 450 μl 1% CuSO4, 450 μl 2% Na-K tartrate,
45 ml 2% Na2CO3
7. Reagent B 3 ml of Folin-Ciocalteu’s phenol reagent,
42ml distilled
water

Protocol

Preparation BSA standard curve

1. 8 plastic tubes were marked and BSA and sodium hydroxide was
added in amounts as mentioned in the table below.

Stock BSA (250 0.3M NaOH Final BSA

μg/ml) Vol in ml concentration
Vol. in ml ( μg/ml)

0 1.0 0

0.1 0.9 25

0.2 0.8 50

0.4 0.6 100

0.6 0.4 150

0.8 0.2 200

0.9 0.1 225

1.0 0 250

2. In a new plastic tube, microsomes were taken and the quantity was
diluted 200 times using 0.3M NaOH. Here 10 μl microsomes were
diluted with 2000 μl or 2.0 ml of 0.3M NaOH.
3. From the above tube 1 ml of the diluted sample was taken into
plastic tubes in duplicate.
4. Then 2 ml reagent A was added to all the tubes and vortex mixed
and left at room temperature for 10 minutes.
5. Then 2 ml of the given reagent B was added to all tubes and then
vortex mixed and left at room temperature for 10 minutes.

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6. Absorbance of standards and samples were measured using

spectrophotometer at 690nm.
7. Standard curves of BSA concentration vs absorbance at 690nm was
plotted to calculate the protein concentration of the microsome
samples (refer table 3 & graph 2).

Results

Formaldehyde calculations
Table 1. Showing standard formaldehyde concentration, absorbance of
formaldehyde at 412nm and corrected values to fit data that in out of range

S.No. Standard Absorbance at Corrected

Formaldehyde 412nm Absorbance

Concentration (Abs at 412-

(nmol/2ml) 0.07)

1 0 0.07 0
2 31 0.105 0.035
3 62 0.176 0.106
4 92 0.212 0.142
5 123 0.255 0.185
6 154 0.339 0.269
7 - - -
8 - - -

Graph 1. Standard curve between formaldehyde concentration against absorbance

at 412nm

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Calculation

Using the line equation y= m x +c

y= 0.001x – 0.008

y+0.0080.001 = x

0.038 + 0.0080,001 = x

x = 46

Similarly, calculating concentration of formaldehyde formed for other

control samples as well as for Phenobarbital induced samples. (refer
table 2.)

Table 2. Showing absorbance and the concentration of formaldehyde formed in the

TCA precipitated samples over 30 minutes of incubation.

Absorbanc Concentration of Absorbance of Concentration of

e of formaldehyde Phenobarbital formaldehyde produced
control produced in control induced in PB induced samples,
samples, using line using line equation y=

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samples equation y= mx + c samples mx + c (nmol/2ml)

(nmol/2ml)

1. 0.038 x = 0.038 + 0.077 X= 0.077 + 0.0080,001

0.0080,001 = 46 = 85
2. 0.041 x= 0.041 + 0.091 X= 0.091 + 0.0080,001
0.0080.001 = 49 = 99
3. 0.056 x =0.056 + 0.112 X= 0.112 + 0.0080,001
0.0080.001 = 64 = 120

4. 0.068 x= 0.068 + 0.139 X= 0.139 + 0.0080,001

0.0080,001 = 76 = 147

5. 0.086 x= 0.086 + 0.157 X= 0.157 + 0.0080,001

0.0080,001 = 94 = 165

6. 0.099 x= 0.099 + 0.173 X= 0.173 + 0.0080,001

0.0080,001 = 107 = 181

Table 3.showing Final BSA concentration and the absorbance of samples at 690nm
and corrected values to fit data that in out of range

S. No. Final BSA Absorbance at Corrected

concentration 690nm Absorbance

( μg/ml) (Abs at 690 –

0.001)

1. 0 0 0
2. 25 0.001 0
3. 50 0.113 0.112
4. 100 0.210 0.209
5. 150 0.331 0.330
6. 200 0.416 0.415
7. 225 0.509 0.508
8. 250 0.620 0.619
Sample 1 (control) - 0.011 -
Sample 2 (control) - 0.12 -
Sample 1 (PB- - 0.015 -
induced)
Sample 2 (PB- - 0.017 -
induced)

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Graph 2. Standard plot of BSA concentration against absorbance of samples at

690nm

Determination of the amount of protein:

– In control samples

Protein concentration using standard BSA curve for control samples,

Average of absorbance values = 0.011 + 0.0122 = 0.0115

y = 0.002 x - 0.025

x = y+0.0250.002

x = 0.0115+0.0250.002 = 18.25 μg/ml (protein concentration from graph)

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This value is then multiplied with dilution factor which was 200

Protein concentration =18.25 x 200

=3650 μg/ml

=3.65 mg/ml

Since we used 50 μl of microsomes so the protein concentration has to be

divided by 1000 and multiplied by 50 to get the final protein concentrations

= 3.65 mg x 50 μl1000 μl

= 0.1825 mg of Protein

Amount of protein in control sample was found to be 0.1825 mg

– In Phenobarbital induced samples

Protein concentration using standard BSA curve for PB - induced samples,

Average of absorbance values = 0.015 + 0.0172 = 0.016

x = y+0.0250.002

x = 0.016+0.0250.002 = 20.5 μg/ml (protein concentration from graph)

This value is then multiplied with dilution factor which was 200

Protein concentration =20.5 x 200

=4100 μg/ml

=4.1 mg/ml

Since we used 50 μl of microsomes so the protein concentration has to be

divided by 1000 and multiplied by 50 to get the final protein concentrations

= 4.1 mg x 50 μl1000 μl

= 0.205 mg of Protein

Amount of protein in control sample was found to be 0.205 mg

Calculation of Velocity
Velocity of the reaction= concentration of formaldehydeduration of the
reaction X protein content(from Lowry'assay)

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Table 4. showing amount of formaldehyde and corresponding reaction

velocities

Formaldehyd Reaction velocity= Formaldehy Reaction velocity=

e formaldehyde de formaldehyde
concentratio concentration30minX protein concentrati concentration30minX protein
n concentration on concentration

in control Or in PB- Or
induced
samples HCHO concentration HCHO concentration
nmol/ml30minX 0.1825 mg samples nmol/ml30minX 0.205 mg
nmol/ml
nmol/ml

46 nmol/ml 8.40 nmol of 85 nmol/ml 13.82 nmol of

formaldehyde/min /mg formaldehyde/min/mg

49 nmol/ml 8.94 nmol of 99 nmol/ml 16.09 nmol of

formaldehyde/min /mg formaldehyde/min/mg

64 nmol/ml 11.68 nmol of 120 19.51 nmol of

formaldehyde/min /mg nmol/ml formaldehyde/min/mg

76 nmol/ml 13.88 nmol of 147 23.90 nmol of

formaldehyde/min/mg nmol/ml formaldehyde/min/mg

94 nmol/ml 17.16 nmol of 165 26.82 nmol of

formaldehyde/min/mg nmol/ml formaldehyde/min/mg

107 nmol/ml 19.54 nmol of 181 29.43 nmol of

formaldehyde/min/mg nmol/ml formaldehyde/min/mg

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Graph 3. Curve of reaction velocity (control sample) against the substrate

concentration[S]. Showing Vmax, Vo and Km.

Vmax -Maximum velocity at which enzyme gets saturated, Vo- Initial velocity, Km- shows
the affinity of the enzyme towards substrate

Graph 4. Curve of reaction velocity (Phenobarbital induced against substrate

concentration. Showing Vmax, Vo and Km.

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Vmax -Maximum velocity at which enzyme gets saturated, Vo- Initial velocity, Km- shows
the affinity of the enzyme towards substrate

Calculation of Vmax and Km from Lineweaver-Burk Plot

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Graph 5. Lineweaver Burk Plot of 1/reaction velocity (Control) against 1/

substrate concentration

Vmax -Maximum velocity at which enzyme gets saturated, Vo- Initial velocity, Km- shows
the affinity of the enzyme towards substrate

Determination of Michaelis Menten constant (Km) for Phenobarbital

induced samples

From the Lineweaver Burk Plot (graph 5), we obtain : y = 0.004 x + 0.060
eq. 1

From the Lineweaver Burk Plot we know that, x = -1/Km value of y=0
eq. 2

So putting values of x and y in eq. 1. We get 0 = 0.004 x -1Km+ 0.06

Rearranging the equation we get, -0.06 = -0.004Km

Km = -0.004-0.06

Km = 0.06 mM

Michaelis Menten’s constant (Km) = 0.066mM

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Determination of Vmax for Phenobarbital induced samples

From the Lineweaver Burk Plot we know that, y = 1Vmax and x=0

So putting values of x and y in eq. 1. We get 1Vmax = 0 + 0.060

Rearranging the equation we get, 1Vmax = 0.06

Vmax = 16.66
nmol/ml/mg

Maximum velocity of the Phenobarbital induced reaction = 16.66

nmol/min/ml

Apparent Vmax for competitive inhibition is same as Vmax so, Vmax’ = 16.66 nmol/
min /ml

Apparent Km cannot be calculated without the Inhibitor concentration and binding constant [Ki] but
for the competitive inhibition apparent Km is lower than the real Km

Graph 6. Lineweaver Burk Plot of 1/reaction velocity (PB-induced) against 1/

substrate concentration

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Vmax -Maximum velocity at which enzyme gets saturated, Vo- Initial velocity, Km- shows
the affinity of the enzyme towards substrate

Determination of Michaelis Menten constant (Km) for Phenobarbital

induced samples

From the Lineweaver Burk Plot (graph 6), we obtain : y = 0.002 x + 0.036
eq. 1

From the Lineweaver Burk Plot we know that, x = -1/Km value of y=0
eq. 2

So putting values of x and y in eq. 1. We get 0 = 0.002 x -1Km+ 0.036

Rearranging the equation we get, -0.036 = -0.002Km

Km = -0.002-0.036

Km = 0.05 mM

Michaelis Menten’s constant (Km) = 0.05mM

Determination of Vmax for Phenobarbital induced samples

From the Lineweaver Burk Plot we know that, y = 1Vmax and x=0

So putting values of x and y in eq. 1. We get 1Vmax = 0 + 0.036

Rearranging the equation we get, 1Vmax = 0.036

Vmax = 27.77
nmol/ml/mg

Maximum velocity of the Phenobarbital induced reaction = 27.77

nmol/min/ml

Apparent Vmax for competitive inhibition is same as Vmax so, Vmax’ = 27.77 nmol/
min /ml

Apparent Km cannot be calculated without the Inhibitor concentration and binding constant [Ki] but
for the competitive inhibition apparent Km is lower than the real Km

Total Cytochrome P450

Value of Total Cytochrome P450 as given to us by demonstrator as 0.632 nmol/mg
(Control) and 0.874 nmol/mg (PB-induced).

But we know, protein obtained from Lowry method as 0.1825mg for control
and 0.205mg for PB induced samples.

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In Control

O.632 = cytchrome P450 per mg of protein from BeerLambert's law Protein

concentration from Lowry'smethod

= cytchrome P450 per mg of protein from BeerLambert's law 0.1825 mg

0.115 nmol = cytchrome P450 in Control (beer lambert’s equation)

In Phenobarbital-induced samples

O.874 = cytchrome P450 per mg of protein from BeerLambert's law Protein

concentration from Lowry'smethod

= cytchrome P450 per mg of protein from BeerLambert's law 0.205 mg

0.179 nmol = cytchrome P450 in Phenobarbital induced samples

( beer lambert’s equation)

Summarization of the results

Control samples Phenobarbital induced

samples

Km 0.06mM 0.05mM

Vmax 16.66 nmol/min/mg 27.77 nmol/min/mg

Amount of Protein 0.1825 mg 0.205 mg

Total cytochrome P450 0.632 nmol/mg 0.874nmol/mg

Discussion :

From the values of total cytochrome P450 in both control (0.632 nmol/mg)
and Phenobarbital (0.874 nmol/mg) it is clear that the Phenobarbital is an
inducer of the enzyme CYP which could be proved by comparing the amount
of total enzyme obtained in both the samples. We can also see a huge
difference in the Vmax of both the samples denoting the high amount of
enzymatic activity in presence of Phenobarbital than in controlled samples.

Km is the concentration at which the clearance is half of the maximum. Km

infers to the affinity of the drug towards enzyme (Rowland & Tozer,1995).

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True Km requires determination of unbound drug concentration at the active

site of enzyme which is not possible in vivo ((Rowland & Tozer,1995).

Graph 7. Comparative graph of the reaction velocities obtained from control

and Phenobarbital induced samples against substrate concentration

The comparative graph above shows that the Km & Vmax of PB induced
(0.05mM) & (27.77nmol/min/mg) is different from the Km & Vmax
(0.06mM) (16.66 nmol/min/mg) of the control. There is big difference in
Vmax of both the sample denoting Phenobarbital as a potent inducer of Cyp
2B1. We got Km of PB induced as 0.05mM which is not significantly lower
than 0.06mM of control. So, we can conclude that it has approximately equal
affinity for its substrate and will reach Vmax/2 faster than the control sample
and Vmax of PB induced samples are much higher than control samples
(difference of 11.11 nmol/min/mg).

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Applying this Km & Vmax value to Pharmacokinetics, we can suggest that

any drug administered along with Phenobarbital will be metabolized faster
and will be eliminated soon from the body than the drug given alone. So the
half life of the drugs will be reduced significantly so large doses or repeated
doses of drug had to be administered in the presence of an inducer to get
the same effect.

From the knowledge of Vmax and Km amount of enzyme to be added to get

required amount to product and time in which product is synthesized could
be determined.

Vmax and Km are constant at a particular pH and temperature. Both Vmax

and km are kinetic parameters of individual enzymes but are not good to
compare enzymes (Roberts et. al., 2003).

Ratio of Vmax/ Km gives the efficiency of the enzyme. But efficiency of the
enzyme is limited by the rate of diffusion of substrate towards the enzyme
(Campbell, 1995).

Absorbance is measured at 450nm after carbon monoxide was passed

because the reduced form of Cytochrome P450 is active form and it
combines with haemeprotein to form complex which has an absorbance
maximum at 450nm, where as oxidized form is inactive and has an
absorbance peak at 420nm.

Extrapolation of the graph was done to fit the values that were out of the
range.

Also to note that some error may be present in the values due human error,
error in pipetting, variation in the incubation time and also cofactor mixture
was insufficient. So the volume was made using buffer. All these factors have
contributed to the error in the values.

The study published by Rowland and Tozer, suggested that the enzyme
induction in the metabolism of those drugs which have high hepatic
extraction ratio will have complications when drug is given orally than when
drug is given intravenously (Rowland & Tozer,1995).

References:

• Campbell, W.H. Lecture notes,1995[available onine]

http://www.bio.mtu.edu/ campbell/401lec12p5.html [accessed on
14 Dec 2009]

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• Chemical carcinogenesis research information centre [available

online]http://toxnet.nlm.nih.gov/ cgi-bin/sis/ search/r?
dbs+ccris:@term+@rn+50-06-6 [accessed on 14 December
2009]

• Denison, M.S, Pandini, A., Nagy, S.R, Baldwin, E.P, Bonati, L.

(2002). Ligand binding and activation of the Ah receptor. Chem.
Biol. Interact. Vol 141 .p1-2.

• Goldstein, A., Aronow, L., Kalman, S. M.(1974).Principles of Drug

action: The basis of Pharmacology . 2nd Ed. John Weiley and sons.
Pg273-282.

• Pharmatutor, 2009 [available online]:

http://www.pharmatutor.org/archive/1/metabolism.html
[accessed on 14 Dec 2009]

• Pubchem compounds [online]. Available from:

http://pubchem.ncbi.nlm.nih.gov/ image/structurefly.cgi?
cid=6009&width=400&height=400 [accessed on 11 Dec 2009]

• Robert, M.K, Garner, D.K., Mayes, P.A., Rodwell, V.W.

(2003).Harper’s illustrated biochemistry. Lang Medical
books/Mcgraw-hill.

• Roberts, R.B., Britten, R. J., Mcclure, F.T.(1961).A model for the

mechanism of enzyme induction. Biophysical journals. Vol 1. p
649-657.

• Rowland, M., Towzer, T.N.(1995). 3rd Ed.Clinical

Pharmacokinetics.Lippincott Williams & Wilkins. Pg 163-188.

• Rang, H.P., Dale, M.M., Ritter, J.M., Moore, P.K.

(2003).Pharmacology. 5th ed. p 557.

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