Drug metabolism in vitro

Aim:

To determine the aminopyrine metabolism in rat liver microsome by measuring the formaldehyde production.

Introduction

Biotransformation of drugs and systhesis of endogenous compounds are largely depends on enzymes of monooxygenase system and several forms of cytochrome P450 (CYP) and NADPH- cytochrome P450 reductase are the main enzymes for phase-? metabolism of exogenous compounds including drugs, whereas phase-?? enzymes include UDP-glucoronosyl transferase, glutathione S-transferase (GST) and N-acetyl and methyltransferase (Sidorova et al., 2004). However, enzyme induction implies that there will be an increase in the rate of its synthesis and an acceleration of its substrate metabolism; translocation of inducer-receptor complex into the nucleus and consequent activation of regulatory elements have been documented as the basic mechanism for the enhanced synthesis of enzyme and for phenobarbital class of inducers the receptor is constitutive androstane receptor (CAR) {Katzung ed. (2004)}.

Pretreatment of rats with phenobarbital stimulate the activity of liver microsomal enzyme, CYP2B1 and consequently the substrate of this enzyme, such as aminopyrine (analgesic and antipyretic drug) metabolism also be enhanced and the duration of its action will be shortened

(Conney et al., 1960). Furthermore, aminopyrin also itself induces CYP2B1 and as a result the rate of its metabolism also be enhanced. It has also been demonstrated that phenobarbital and aminopyrin both increase the synthesis of drug metabolizing enzyme (CYP2B sub family) in the liver microsomes and stimulate overall liver protein synthesis ( Conney et al., 1960) that is also evident from the higher amount of cytocrome P450 in phenobarbital induced sample in this experiment.

By using of aminopyrine as substrate and by measuring formaldehyde as product rat-liver microsomal N-demethylase activity was first studied by Ernster and Orrenius (1965) and the location of systems that involves in the oxidative N-demethylation of aminopyrine, is in hepatic microsomes was detected by La Du et al. (1955) { Gram et al., 1967}. However, in this experiment metabolism of aminopyrin, by measuring (using the Nash reagent) the formed formaldehyde by N-demethylation in hepatic microsomal preparation from control and phenobarbital treated rats is determined and compared. Substrate optimum, Km and Vmax for aminopyrin metabolism is also studied in controlled and PB-treated rat liver.

Aminopyrine metabolism ( Imaoka et al.,1988),

Aminopyrine N-demethylase MAA (monomethylaminoantipyrine) + formaldehyde.

N-demethylase AA (aminoantipyrine) + formaldehyde.

Methods and Materials

Animals

Male Sprague Dawley rats (250 - 350g) were used. Phenobarbital sodium was administered according to the following schedule: 80mg/kg in physiological saline for 3 days or with saline alone for 3 days and on the fourth day the animals were killed by cervical dislocation, livers were isolated, weighed and microsomes were prepared.

Preparation of Microsome

All buffers were pre-chilled to 4°C and the isolation procedure carried out on ice. The livers were excised into washing buffer (1.15% KCl in 0.01M Tris-HCl, pH 7.4) then homogenised in 4 x liver weight of homogenisation buffer (1.15% KCl, 15% Glycerol in 0.1M Tris-HCl, pH 7.4) using a motor driven Potter-Elvehjem pestle and tube. Afterwards, the homogenates were then centrifuged at 9,000g for 20 min at 4°C. The supernatant was then centrifuged at 180,000g for 60 min at 4°C. The resultant supernatant (cytosolic fraction) was discarded and the pellets re-suspended in homogenising buffer and centrifuged at 180,000g for a further 60 min at 4°C.The pellets were then re-suspended in 0.5 x liver weight of storage buffer (20% Glycerol in 0.1M Tris-HCl, pH 7.4) and stored frozen at -80°C.

Measurement of Total Cytochrome P450

Microsomes were diluted 50-fold (20ml + 980 ml Tris-HCl buffer, pH 7.4) and 1ml pipetted into two matched cuvettes. A baseline spectrum was obtained between wavelengths 390-500nm using a scanning spectrophotometer. Carbon monoxide gas was gently bubbled for 30 seconds through the sample cuvette and then a few grains of sodium dithionite were added to both sample and reference cuvettes. A difference spectrum was obtained between the mentioned wavelengths.

The absorbance value for the 450nm peak was determined and multiplied by 50 to take account of the dilution factor. By using Beer - Lambert equation the cytochrome P450 concentration (in mM) was calculated and by applying this concentration and other concentration (mg/ml) obtained by Lowry method the amount of cytochrome P450 per mg of protein was measured.

Determination of the Kinetic Constants for Aminopyrine N-Demethylation

Formaldehyde solution (37% (w/v), cofactor buffer mix (containing 0.25mM NADP+, 2.5mM DL-isocitric acid, 0.6 Units isocitrate dehydrogenase, 5mM MgSO4, 0.1M phosphate buffer pH 7.4), 5mM aminopyrine ,40mM aminopyrine, 20% (w/v) trichloroacetic acid (TCA), Nash reagent (containing, 45g ammonium acetate, 0.6 ml acetylacetone and 0.9 ml glacial acetic acid dissolved in distilled water to a final volume of 100 ml) were used as chemical reagents.

Preparation of incubation sample tubes

Table 1: Preparation of sample tubes both in control and PB induced samples
(8 sample tubes for each)

Sample tube number		1	2	3	4	5	6	7	8
Vol. cofactor mix	ml	1.85	1.85	1.85	1.85	1.85	1.85	0	0
Vol. buffer	ml	0	0	0	0	0	0	1.85	1.85
Vol. microsomes	ml	50	50	50	50	50	50	50	50
Vol. water	ml	75	50	0	75	50	0	0	100
Table 2 : Volume and stock concentration of aminopyrine
Vol. 5mM aminopyrine	ml	25	50	100	0	0	0	0	0
Vol. 40mM aminopyrine	ml	0	0	0	25	50	100	100	0
Final aminopyrine concentration (S)	mM	0.0625	0.125	0.25	0.5	1	2	2	0

Microsomal incubations

By using a stopwatch the samples were distributed at 30-second intervals and initiation and termination of the reaction was done according to the Table 3: Timing schedule for reaction initiation and termination.

Sample tube no.	Pre-incubation start time	Aminopyrine addition time (initiation)	TCA (20% w/v) addition time (termination)
Tube 1	0 sec	4.0 min	34 min
Tube 2	30 sec	4.5 min	34.5 min
Tube 3	1.0 min	5.0 min	35 min
Tube 4	1.5 min	5.5 min	35.5 min
Tube 5	2.0 min	6.0 min	36 min
Tube 6	2.5 min	6.5 min	36.5 min
Tube 7	3.0 min	7.0 min	37 min
Tube 8	3.5 min	N/A	37.5 min

Reaction was initiated by adding aminopyrin according to table 2 and was terminated by adding 0.5 ml 20% TCA at the end of 30 minutes. Once the reaction had been terminated, samples were centrifuged to precipitate the microsomal protein.

Preparation of standard formaldehyde solution

0.0037%. w/v was prepared from 37% w/v formaldehyde solution. Final concentration was expressed as µM/ml in the following way-

100 ml contains .0037 gm of formaldehyde and when 30 gm formaldehyde is present in 1000ml then the strength of solution will be = 1M = 10-3µM. So, 0.0037 gm of HCHO in 1 ml will produce the final strength of 12.33 × 10?4 µM and that can be expressed as 12.33 × 10?4 µM/ml.

Preparation of formaldehyde standard curve

From this diluted stock (0.0037%), a formaldehyde standard curve was prepared, in plastic tubes according to table 4 and 0.5ml of 20% TCA was added to each tube.

Table 4: Preparation of formaldehyde standard curve

Standard no.	S1	S2	S3	S4	S5	S6
Vol. standard HCHO (ml)	0	25	50	75	100	125
Vol. Buffer (ml)	2.0	1.975	1.95	1.925	1.9	1.875
nmol of HCHO/2ml	0	31	62	92	123	154

Nash reaction

1.5ml Nash reagent was pipette into each tube (standard and samples) and 1.5 ml of the standards and supernatant from each TCA-precipitated incubation sample was added to each of these tubes; after vortex mixing and placing them into a 60°C water bath and cooling the absorbance of the solutions by using distilled water was read out at wavelength of 412nm.

Determination of Protein by the Lowry Method

Reagents used- 0.3M sodium hydroxide, 250mg bovine serum albumin (BSA) per ml in 0.3M sodium hydroxide, 1% (w/v) CuSO4, 2% (w/v) Na-K tartrate, 2% (w/v) Na2CO3 and Folin-Ciocalteu's phenol.

Preparation of BSA standard curve

Stock BSA (250mg/ml) volume in ml	0.3M NaOH volume in ml	Final BSA Concentration (mg/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

Microsome samples were diluted by 200-fold in 0.3M NaOH and to do it 15µl               microsomes was mixed with 2.985 ml 0.3M NaOH . 1.0 ml of the diluted microsome sample was taken into plastic tubes in duplicate.

Preparation of Reagent A was done by mixing 450 ml of 1% (w/v) copper sulphate, 450 ml of 2% (w/v) sodium potassium tartrate and 45 ml of 2% (w/v) sodium carbonate with each other and 2ml of reagent was added to the all tubes.

Preparation of Reagent B was done by mixing 3 ml of Folin-Ciocalteau's phenolic reagent and 42 ml of distilled water with each other and 2 ml of this was further added to the each tube. Absorbance of the standards and samples using a spectrophotometer at wavelength 690nm was measured.

Determination of protein concentralion in microsomal samples

Plotting the Standard curve of BSA concentration (µg/ml) Vs absorbance at 690 nm

Figure 1 : Bovine serum albumin standard curve

Table 5: Absorbance value of microsomal samples

	Control absorbance	PB induced absorbance
	0.018	0.032
	0.022	0.036
Average	0.02	0.034

By using the above BSA standard curve, y = .0024x - 0.0271

Here, y = absorbance

x = concentration of protein So, concentration of protein will be in Control = 0 .00271+0.02/0.024 = 19.63 µg/ ml And similarly, in PB induced concentration of protein = 25.46 µg/ml.

By, considering the dilution factor, as for our convenience initially the sample was diluted 200 times. So, to get the actual protein concentration it is needed to be multiplied by 200.

Therefore,

In control, actual protein concentration in microsomal sample will be = 19.63 × 200 µg/m

= 3926 µg/ml.

In PB induced, actual protein concentration in microsomal sample will be = 25.46 × 200 µg/ml.

= 5092 µg/ml.

So, concentration in mg/ml in control, will be =3926/1000 = 3.926 mg/ml

And in PB induced, will be = 5092/1000 = 5.092 mg/ml

Results and discussion

Calculation of the amount (nmol) of CYP450 per mg of protein

Amount (nmol) of cytochrome P450 (CYP450) per mg of protein =

Total CYP450 concentration (µM) obtained using Beer-Lambert equation x 1000

Total protein concentration (mg/mL) obtained using Lowry equation

Therefore,

The amount of CYP450 in control = 0.632 nmol/mg/mL and

The amount of CYP450 in PB-induced = 0.874 nmol/mg/mL.

The amount of cytochrome P450 was found higher in PB induced sample of microsome than the control which indicates that phenobarbital enhanced the synthesis of CYP isozymes in rat liver microsome. Furthermore, higher protein concentration (5.092 mg/ml) in PB induced samples obtained in Lowry method suggests that overall liver protein synthesis was increased due to the inducing effect.

Plotting of formaldehyde standard curve value i.e. absorbance at 412nm versus nmol formaldehyde/2mL

Fig 2: Formaldehyde standard curve

The graph was obtained by measuring the absorbance of the nmol HCHO/2mL in six tubes (S1 to S6) according to the table 4. By subtracting the value of absorbance at zero nmol/2mL concentration of formaldehyde from all concentrations (0, 31, 62, 92, 123, 154) an equation of straight line was found which passed through the origin.

Calculation of nmol of formaldehyde formed in the TCA-precipitated samples over the 30min incubation time from the HCHO standard curve

By appling the equation from figure 2 ,the nmol of HCHO formed in TCA- precipitated samples was calculated. Using spectrophotometer measurement of unknown samples absorbance both in control and PB induced were done and their corresponding values of concentration (calculated from figure 2 equation) have been showed in table 6.

Table 6: Amount of formaldehyde formed in control and PB induced samples

Control		Phenobarbital induced
Absorbance (y)	Conc (nM)/2mL= (y+0.0097)/0.0018	Absorbance (y)	Conc (nM)/2mL= (y+0.0097)/0.0018
0.009	10.39	0.045	30.39
0.012	12.06	0.059	38.17
0.027	20.39	0.080	49.83
0.039	27.06	0.107	64.83
0.057	37.06	0.125	74.83
0.070	44.28	0.141	83.72

The absorbance values in table 6 is the value after substracting the tube 8 absorbance value  as it did not contain any co-factor mix and aminopyrine (Table 1 and 2). Again, according to the table 1 and 2, tube 7 only contained aminopyrin and used only to verify the effect of aminopyrine without co-factor. Therefore, tube 7 is also not included in table 6. However, increased trend of formaldehyde concentration both in control and PB induced samples, from tube 1 to tube 8  is due to progressive increase in  aminopyrine concentration from tube 1 to tube 8 (Table 2) and higher HCHO production  in PB induced samples than controlled is an evidence of increased metabolism in aminopyrine in PB induced samples. It might be due to enhancement of N-demethylase activity in rat liver microsome was occurred through the phenobarbital induction which subsequently increase the demethylation of aminopyrine as well as its metabolism (Imaoka et al.,1988).

Calculation of the reaction velocity (v) for each sample i.e. nmol HCHO formed/minute/mg protein

Table 7: Amount of Formaldehyde formation in thirty minutes in Control and PB induced samples

Control		Phenobarbital induced
Concentration (nM/2mL)[Cc]	Amount HCHO over 30 mins (nM/min/mL)= [Cc]/(2x30)	Concentration (nM/2mL)[CPB]	Amount HCHO over 30 mins (nM/min/mL)= [CPB]/(2x30)
10.39#	0.17	30.39#	0.51
12.06	0.20	38.17	0.64
20.39	0.34	49.83	0.83
27.06	0.45	64.83	1.08
37.06	0.62	74.83	1.25
44.28	0.74	83.72	1.40

Reaction velocity [V] (nM/min/mg) = Amount of HCHO over 30 mins (nM/min/mL)

Protein content from Lowry assay (mg/mL)

# Eg. In control, [V] (nM/min/mg) for 10.39nM/2mL concentration = 0.17/3.926 = 0.04 nM/min/mg and in case of PB induced, [V] for 30.39nM/2mL concentration = 0.51/5.092 = 0.1 nM/min/mg. The subsequent values for the corresponding concentration have been presented in table 8.

Table 8: Reaction velocity in Control and PB induced sample

Reaction velocity [V] (nM/min/mg)
Control	Phenobarbital induced
0.04	0.10
0.05	0.12
0.08	0.16
0.11	0.21
0.15	0.24
0.18	0.27

Table 8 shows the higher reaction velocity in PB-induced microsomal samples than in control. As PB induced sample contains higher CYP 450 due to inducing effect of Phenobarbital, so more free enzyme will be available for catalyzing substrate (aminopyrine). As a result higher reaction velocity will be observed in PB induced samples

Construction of plots[V] versus [S]: Here [V] is the reaction velocity and [S] is the aminopyrine concentration in mM.

Figure 3: Michaelis-Menten plot of reaction velocity of aminopyrine versus it concentration

1/[V] versus 1/[S] (the Lineweaver-Burk plot).

i. Calculation of apparent Km and Vmax values from the Lineweaver-Burk plot

From the Lineweaver-Burk plot as shown in above Figure the (-1/Km) and (-1/Vmax) values for control and Phenobarbital induced microsome has been showed in arrow.

For control [Km is expressed = Km(c) and Vmax is expressed =Vmax(c)]

Here, -1/Km (c) ˜ - 4.5 and 1/Vmax (c) ˜ 6.0 nmol/ min/ mg

Therefore, Km (c) ˜ 0.22 and Vmax (c) ˜ 0.17 nmol/ min/ mg

For Phenobarbital induced [ Km is expressed =Km(PB) and Vmax is expressed =Vmax(PB)]

Here, -1/Km (PB) ˜ -10.0 and 1/Vmax (PB) ˜ 4 nmol/ min/ mg

Therefore, Km (PB) ˜ 0.10 and Vmax (PB) ˜ 0.25 nmol/ min/ mg

Table 9: Apparent Km and apparent Vmax for formaldehyde production in Control and PB induced samples.

Control					Phenobarbital induced
Apparent Km(c)		Apparent Vmax(c)			Apparent Km(PB)		Apparent Vmax(PB)
0.22		0.17 nmol/min/mg			0.10		0.25 nmol/min/mg

Table 09 shows that the maximum reaction rate Vmax in case of PB-induced microsomal sample 0.25nmol/min/mg which is much higher than that of control indicative of higher reaction velocity of aminopyrine in PB-induced microsomal sample. It can be explained by the inducing effect of Phenobarbital which consequently increases the demethylation of aminopyrine as well as metabolism in PB- induced microsomal sample. On the contrary, absence of inducer in control will produce no peak in metabolism rather than the normal metabolism.

Whether the rate of formation of product will be affected by the availability of substrate or not can be predicted from the Km of an enzyme. Table 9 shows that in PB induced samples the apparent Km value is much lower than the control. It can be said that due to higher amount of available enzymes in PB induced sample causes more formation of enzyme-substrate complex and then less substrate will be available for further reaction.

Conclusion:

Probes into the regulating factors of drug metabolising systems are often carried out in small animal species and most of these studies have done in vitro enzyme preparation. However, the lack of commodious way in vivo procedure has hampered advances in this area; many of the striking effects demonstrated by use of microsomal systems still needs to be affirmed in vivo conditions, while factors like blood flow or absorption rate may alter or even reverse the measurement of in vitro effect ( Houston et al., 1981).

References :

Houston, J.B., Lockwood, G. and Taylor, G (1981). Aminopyrine Demethylation Kinetics: Use of metabolite exhalation rates as an index of enhanced mixed-function oxidase activity in vivo. Drug metabolism and disposition.9, 449-455.

Sidorova, Y.A and Grishanova, A.Y (2004). Dose- and Time-dependent effects of Menadione on Enzymes of Xenobiotic Metabolism in Rat liver. Bulletin of Experimental Biology and Medicine. 137, 260-264.

Katzung,ed.(2004). Basic &Clinical Pharmacology.9th Ed. USA: McGraw-Hill Companies.

Conney, A. H., Davison, C., Gastel, R. and Burns, J. J. (1961). Adaptive increase in drug metabolizing enzymes induced by Phenobarbital and other drugs. The journal of pharmacology and experimental therapeutics. 130, 1-8.

Gram, T.E., Wilson, J.T and Fouts, J.R. (1968). Some characteristics of hepatic microsomal systems which metabolize aminopyrine in the rat and rabit. JPET. 159, 172-181.

Imaoka S,Inoue K,Funae Y. (1988). Aminopyrine metabolism by multiple forms of cytochrome P-450 from rat liver microsomes: simultaneous quantitation of four aminopyrine metabolites by high-performance liquid chromatography. Arch Biochem Biophys.265(1), 159-70.