11-12-09 report

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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).

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)

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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

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

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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

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.

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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.

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.

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Sample tube number 1 2 3 4 5 6 7 8

Vol. water (μl) 75 50 0 75 50 0 0 100

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

Vol. of microsomes (μl)

50 50 50 50 50 50 50 50

Vol of cofactor mixture (ml)

1.85 1.85 1.85 1.85 1.85 1.85 0 0

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 8Vol of 5mM Aminopyrine (μl)

25 50 100 0 0 0 0 0

Vol of 40mM Aminopyrine (μl)

0 0 0 25 50 100 100 0

4. All tubes were vortex mixed and were placed in water bath again.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 curve7. 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 S6Vol of standard formaldehyde (μl)

0 25 50 75 100 125

Vol of buffer (ml) 2.0 1.975 1.95 1.925 1.9 1.875nmol of HCHO/2ml 0 31 62 92 123 154

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

Nash Reaction9. 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 M2. BSA (bovine serum albumin) 250 g/ ml in 0.3M NaOH

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 μg/ml)Vol. in ml

0.3M NaOHVol in ml

Final BSA concentration( μg/ml)

0 1.0 00.1 0.9 250.2 0.8 500.4 0.6 1000.6 0.4 1500.8 0.2 200

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0.9 0.1 2251.0 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.

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 Formaldehyde Concentration (nmol/2ml)

Absorbance at 412nm Corrected Absorbance(Abs at 412- 0.07)

1 0 0.07 02 31 0.105 0.0353 62 0.176 0.1064 92 0.212 0.1425 123 0.255 0.1856 154 0.339 0.2697 - - -8 - - -

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Graph 1. Standard curve between formaldehyde concentration against absorbance at 412nm

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

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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.

Absorbance of control samples

Concentration of formaldehyde produced in control samples, using line equation y= mx + c (nmol/2ml)

Absorbance of Phenobarbital induced samples

Concentration of formaldehyde produced in PB induced samples, using line equation y= mx + c (nmol/2ml)

1. 0.038x =

0.038+0.0080,001

= 46 0.077

X= 0.077+0.008

0,001 = 85

2. 0.041 x=0.041+0.0080.001

= 490.091

X= 0.091+0.008

0,001 = 99

3. 0.056x ¿

0.056+0.0080.001

= 640.112

X= 0.112+0.008

0,001 = 120

4. 0.068x¿

0.068+0.0080,001

= 760.139

X= 0.139+0.008

0,001 = 147

5. 0.086x¿

0.086+0.0080,001

= 940.157

X= 0.157+0.008

0,001 = 165

6. 0.099x¿

0.099+0.0080,001

= 1070.173

X= 0.173+0.008

0,001 = 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 concentration( μg/ml)

Absorbance at 690nm Corrected Absorbance (Abs at 690 – 0.001)

1. 0 0 02. 25 0.001 03. 50 0.113 0.1124. 100 0.210 0.2095. 150 0.331 0.3306. 200 0.416 0.4157. 225 0.509 0.5088. 250 0.620 0.619Sample 1 (control) - 0.011 -Sample 2 (control) - 0.12 -Sample 1 (PB-induced) - 0.015 -Sample 2 (PB-induced) - 0.017 -

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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+0.012

2 = 0.0115

y = 0.002 x - 0.025

x = y+0.025

0.002

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x = 0.0115+0.025

0.002 = 18.25 μg/ml (protein concentration from graph)

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

= 3.65mgx 50μ l

1000μ l

= 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+0.017

2 = 0.016

x = y+0.025

0.002

x = 0.016+0.025

0.002 = 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

= 4.1mgx 50μ l

1000μ l

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= 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 formaldehyde

durationof the reactionX protein content (¿ Lowr y ' assay)

Table 4. showing amount of formaldehyde and corresponding reaction velocities

Formaldehyde concentration in control samples nmol/ml

Reaction velocity= formaldehyde concentration

30min X protein concentrationOr

HCHOconcentration nmol /ml30min X0.1825mg

Formaldehyde concentration in PB-induced samples nmol/ml

Reaction velocity= formaldehyde concentration

30min X protein concentrationOr HCHOconcentration nmol /ml

30min X0.205mg

46 nmol/ml 8.40 nmol of formaldehyde/min /mg 85 nmol/ml 13.82 nmol of formaldehyde/min/mg

49 nmol/ml 8.94 nmol of formaldehyde/min /mg 99 nmol/ml 16.09 nmol of formaldehyde/min/mg

64 nmol/ml 11.68 nmol of formaldehyde/min /mg 120 nmol/ml 19.51 nmol of formaldehyde/min/mg

76 nmol/ml 13.88 nmol of formaldehyde/min/mg 147 nmol/ml 23.90 nmol of formaldehyde/min/mg

94 nmol/ml 17.16 nmol of formaldehyde/min/mg 165 nmol/ml 26.82 nmol of formaldehyde/min/mg

107 nmol/ml 19.54 nmol of formaldehyde/min/mg 181 nmol/ml 29.43 nmol of formaldehyde/min/mg

Graph 3. Curve of reaction velocity (control sample) against the substrate concentration[S]. 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

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 = 1

Vmax and x=0

So putting values of x and y in eq. 1. We get 1

Vmax = 0 + 0.060

Rearranging the equation we get, 1

Vmax = 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 = 1

Vmax and x=0

So putting values of x and y in eq. 1. We get 1

Vmax = 0 + 0.036

Rearranging the equation we get, 1

Vmax = 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 P 450 permgof protein ¿BeerLamber t' s law ¿Protein concentration ¿

Lowr y ' smethod ¿

¿cytchrome P450 permgof protein ¿BeerLamber t' s law ¿0.1825mg

0.115 nmol = cytchrome P 450 in Control (beer lambert’s equation)

In Phenobarbital-induced samples

O.874 =

cytchrome P 450 permgof protein ¿BeerLamber t' s law ¿Proteinconcentration ¿

Lowr y ' smethod¿

¿cytchrome P450 permgof protein ¿BeerLamber t' s law ¿0.205mg

0.179 nmol = cytchrome P 450in Phenobarbital induced samples ( beer lambert’s equation)

Summarization of the results

Control samples Phenobarbital induced samples

Km 0.06mM 0.05mMVmax 16.66 nmol/min/mg 27.77 nmol/min/mgAmount of Protein 0.1825 mg 0.205 mgTotal 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). True Km requires

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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).

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

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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]

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.

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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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