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Study of coumarin metabolism by human liver microsomes using capillary electrophoresis
ANALmcA CHIMICA
ACTA Analytica &mica Acta 310 (1995) 101-107
Study of coumarin metabolism by human liver microsomes using capillary electrophoresis Brian Deasy a, Declan P. Bogan b, Malcolm R. Smyth a**, Richard O’Kennedy b, Uwe Fuhr ’ a School of Chemical Sciences, Dublin City University, Dublin 9, Ireland b School of Biological Sciences, Dublin City University, Dublin 9, Ireland ’ Department of Clinical Pharmacology, University Hospital, D-60590 Frankfurt am Main, Germany Received 17 January 1995; revised 8 February 1995; accepted21 February 1995
Abstract A method for the rapid determination of 7-hydroxycoumarin, based on separation by capillary electrophoresis (CE), with UV absorbance detection at 210 nm, was applied to a study of the metabolism of coumarin in human liver microsomes. The linear detection range for 7-hydroxycoumarin was O-SO pg/ml, while the limit of detection was 1 pg/ml. Human liver microsomes from 5 patients were used in metabolic studies and the profiles clearly indicated that there is inter-individual variability in coumarin 7-hydroxylase activity. The CE method allowed the investigation of NADP+/NADPH cofactor reactions involved in coumarin metabolism.The CE method was also compared to a liquid chromatographic method for the analysis of the metabolite and it was found that the results did not differ but the CE method was more rapid and needed no sample preparation. The very short time between sampling and analysis makes the method suitable for automated sample transfer and measurement. Keywords: Electrophoresis; Coumarins; Liver microsomes
1. Introduction The metabolism of coumarin (SH) in the human body can be described by reaction 1 (Fig. 1) NADPH+H++02+SH P450 + NADP+
+ H,O + SOH
(1)
where P450 is a cytochrome P450 enzyme and SOH is 7-hydroxycoumarin. NADPH (nicotinamide
* Corresponding author.
adenine dinucleotide phosphate) is needed to donate 2 electrons to the heme iron of cytochrome P450 [l]. Microsomal preparations from human liver contain cytochrome P450 isoforms and have been used to study the metabolic pathways of drugs [2-51. NADPH is regenerated by the use of glucosed-phosphate dehydrogenase and is thus recycled. Coumarin, a naturally occurring plant constituent, has been used in the treatment of cancer [6] and oedemas [7]. After administration, in humans, coumarin is quickly metabolised to 7-hydroxycoumarin by a specific cytochrome P450 (P45OCoh) in the liver [8] and therefore may be used to monitor
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COUMARIN
p450,
Chimica Acta 310 (1995) 101-107
HoyJy 'I-HYDROXYCOUMARlN
Fig. 1. Conversion of coumarin to 7-hydroxycoumarin.
NADPH and NADP+ migration. The electrolyte was prepared daily by mixing 0.02 M K,HPO, and 0.005 M KH,PO, in deionised water. Coumarin (2.05 X 10m4 M in 0.1% methanol: 99.9% ultra pure water) was donated by Schaper and Brummer. 2.2. Tissue preparation
activity of the isoform. It has also been suggested that 7-hydroxycoumarin is a prodrug for coumarin [9] and it is currently being investigated in clinical trials for its effectiveness in cancer treatment [lo]. Capillary electrophoresis (CE) is a recent addition to the tools available in the analytical and research laboratory, and its range of applications is dramatically increasing, e.g., for the analysis of drugs, and biological macromolecules [ll-151. The metabolism of coumarin has been monitored in the past by liquid chromatography (LC) [17,18]. However, one needs to remove the protein in the sample to protect the LC system from injection to detection. In a recent paper we have described a CE method for the determination of 7-hydroxycoumarin in urine and serum [16]. This new paper describes the application of this method to follow the metabolism of coumarin directly in the presence of liver microsomal preparations without any sample preparation. Furthermore, the analysis time for the metabolite is considerably reduced in comparison to LC methods [17,18]. The method applied also allowed the monitoring of NADPH and NADP+ levels present, and to assess their utilisation in the metabolic system.
2. Experimental 2.1. Chemicals 7-Hydroxycoumarin, NADPH, NADP+, D-glucose-6-phosphate and glucosed-phosphate dehydrogenase were purchased from Sigma (St. Louis, MO). KH a PO, and K, HPO, were obtained from Kiedel-de Haen (Hannover). 7-Hydroxycoumarin standards were prepared from a 1 mg/ml stock solution in methanol (HPLC grade, Labscan, Dublin): deionised water (10:90, v/v> by dilution in deionised water. The electrolyte solution used when monitoring metabolite formation was 0.025 M phosphate buffer, pH 7.0. The pH was adjusted to 7.5 when monitoring
The five donors of human microsomes were patients either with carcinoma of the colon or small intestine and a single liver metastasis, or patients with primary liver tumours, who all underwent partial hepatectomy. Portions of macroscopically healthy liver, taken from the excised lobe, were chilled immediately in 1.15% KC1 (0” C) and were then frozen in liquid nitrogen within a few minutes. Microsomes were obtained by differential centrifugation using the sucrose method [19]. The characteristics of the donors are summarised in Table 1. The protein concentrations of these microsome preparations were calculated using the bicinchoninic acid (BCA) assay (Pierce).
2.3. Incubation solution The concentrations of the NADPH-generating system in all incubations were 4 U/ml glucosed-phosphate dehydrogenase, 2 mg/ml p-glucosed-phosphate, and 1.2 mM NADP+. This was higher than that required to maintain maximum metabolic rates under the chosen in vitro conditions. The coumarin (substrate) concentration in the incubation solutions was always 1.6 X low4 M. For the incubations 195 ~1 of a coumarin standard (30 pg/ml, prepared in phosphate buffer, pH 7.5, 25 mM>, 10 ~1 of D-glucose-6-phosphate (50 mg/ml), 15 ~1 of NADP+ (20 mM) and 5 ~1
Table 1 Details of liver donors Liver
Donor sex
Donor age (years)
Smoking status
P13 P14 P17 P18 Rl
F F F F M
64 48 54 44 54
NS NS S NS NS
B. Deasy et al./Analytica Chimica Acta 310 (1995) 101-107
glucose-6-phosphate dehydrogenase were mixed together in an Eppendorf tube to give final concentrations as above. The reaction was started by the addition of 25 ~1 of microsomal suspension (the microsomal preparations are called P13, P14, P17, P18 and Rl), resulting in a final protein concentration of about 2 mg/ml. Following mixing, the open Eppendorf tube was transferred immediately into the incubation bath (37” Cl, and a 20 ~1 sample was taken and analysed. The metabolite formation was monitored over a 2 h period, taking samples at 0, 7, 14, 20, 30, 45, 60, 90 and 120 min, respectively. These samples were analysed using a Beckman capillary electrophoresis instrument (P/ACE System 2050). In the case of analysis by LC, the incubation was first stopped by the addition of 50 ~1 of ice-cold trichloroacetic acid (20% w/v> and centrifuged (low speed) before injection. The LC analysis was carried out according to Egan and O’Kennedy [20]. 2.4. NADP ‘/NADPH
ratio study
103
mal preparations, the concentration being calculated from a plot of absorbance versus standard concentration ( pg/ml). The linear detection range for 7-hydroxycoumarin was O-50 pg/ml and the limit of detection was 1 pg/ml.
3. Results and Discussion
3.1. Development of the CE separation The CE method developed for monitoring 7-hydroxycoumarin in plasma and urine [15] was adapted for studying the metabolism of coumarin by human liver microsomes. When interested in NADP+ (2 mM) and NADPH, a pH of 7.5 was required as these compounds exhibit a very poor peak shape in pH 7.0 electrolyte (phosphate buffer, 25 mM). The absorbance of coumarin and 7-hydroxycoumarin could both be monitored with good sensitivity at 210 nm. Fig. 2 shows an overlay of the typical electropherograms at 0 min and after 45 min for the metabolism
For this study, the concentration of glucosedphosphate dehydrogenase was reduced to 0.1 U/ml. The remainder of the incubation mixture was as above. The study was carried out in the presence and in the absence of coumarin, or the microsomal preparation. 2.5. Capillary electrophoresis separation The capillary used was a 27 cm X 50 pm fused silica column (Beckman), with a capillary to detector distance of 19.3 cm. The preparation step for priming of the capillary was a 1 min rinse with 0.1 M sodium hydroxide, followed by a 1 min rinse with electrolyte solution (0.025 M phosphate buffer, pH 7.0 or pH 7.5, depending on the separation required). The sample was applied to the capillary by a 3 s pressurised injection (0.5 p.s.i.1 and separation achieved with an applied voltage of 20 kV (rise time 0.2 min) at 25” C. Typical running current was 100 PA. The resultant separation was monitored at 210 nm with a fixed wavelength detector using Beckman System Gold” software. Concentrations of 7-hydroxycoumarin were calculated from the mean of three standard curves prepared by spiking standards of 7-hydroxycoumarin into three different microso-
0.03
,E s Lu
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co B
d
2
0.01
0.00 0.0
0.6
1.0
1.6
2.0
2.6
9.0
2.5
Time (minutes) Fig. 2. Overlay of electropherograms showing separation of (A) all neutral compounds including coumarin, (B) 7-hydroxycoumarin, (C) microsomes, (D) NADP+ and (El NADPH, at 0 min (solid line) and at 45 min (dashed line) monitored at 210 nm using 25 mM phosphate buffer at pH 7.5.
104
B. Deasy et al./Analytica
Chimica Acta 310 (1995) 101-107
0.016
jl
c
E z Z 8 5
0.011
?2 % 9
J 0.006
I
0.6
I 1.0
Time
I
c
I
1.2
1.4
(mlnutes)
Fig. 3. Overlay of electropherograms showing separation of (A) all neutral compounds including coumsrin, (B) 7-hydroxycoumarin, (C) microsomes, at 0 min (solid line) and at 45 min (dashed line) monitored at 210 nm using 25 mM phosphate buffer at pH 7.0.
it is possible to say that all the compounds that have longer migration times than coumarin are negatively charged and hence migrate more slowly than coumarin. 7-Hydroxycoumarin is more negatively charged than coumarin due to the hydroxyl group at the 7-position. This hydroxyl group is partially ionised at this PH. The microsomes migrate more slowly than the 7-hydroxycoumarin and are seen as a broad peak due to the different types of proteins in the microsomal suspensions. NADP+ has a structure with four negative charges and a positively charged nitrogen associated with it. This suggests an overall charge of 3 negative charges. This high overall negative charge explains the slow migration of NADP+. The NADPH molecule has a similar structure to NADP+ but the positive charge on the nitrogen is no longer present. As a result, this more negatively charged compound has the slowest migration rate of all the compounds. Control incubations included an absence of (A) coumarin, (B) microsomes, and (C) the NADPH regeneration system (Fig. 3). Coumarin was not quantifiable under these conditions as other unknown reactions were occurring producing neutral compounds which co-migrate with coumarin. The neutral compound produced was dependent on the NADPH reaction as observed with control (A). 3.2. Coumarin metabolism profiles
incubation for the P18 microsomal preparation. The metabolite, 7-hydroxycoumarin, migrates at 0.9 minutes and its increase can be followed under the conditions outlined under Experimental above. However, it is not well separated from the broad peak of the microsomal preparations at pH 7.5 and thus it must be monitored at pH 7.0 (Fig. 31 to resolve the compounds. It can be seen from Fig. 2 that NADP+ and NADPH are well separated and quantifiable at this pH. The use of a micellar-based electrolyte solution was also assessed, but there was no benefit found in utilising such a system for the determination of 7-hydroxycoumarin. A neutral marker (benzamide) was run under the conditions above, and it was found to co-migrate with coumarin, thus suggesting that coumarin is behaving as a neutral molecule at this pH. This is not surprising when the structure is considered (Fig. 1). By knowing the migration time of the neutral marker
Microsome preparations from 5 different patients were used in metabolism studies. Incubations were carried out in triplicate. Fig. 4 shows a plot of concentration of 7-hydroxycoumarin (nmol/ml/mg protein f SD.), versus time for each of the five metabolic studies. It shows that each of the microsomal preparations has a different coumarin metabolism profile; this is in agreement with results from other groups [ 171. In fact, the inter-individual variability of human coumarin 7-hydroxylase activity in liver microsomes is very large, and can be up to 100 fold [21]. Although the errors in concentration calculations are relatively high, the results correspond to microsomal drug metabolism results from other researchers [2]. Fig. 5 shows the comparison of using LC against CE for the analysis of the metabolite formation. It can be seen that there is no significant difference between the results from both methods.
B. Deasy et al./Analytica Chimica Acta 310 (1995) 101-107
LC is the common method used for this analysis of metabolites in cytochrome P450 studies 12-51 and also for the quantification of 7-hydroxycoumarin. The LC methods involve stopping the enzyme reactions by precipitating out the protein to minimise degradation of the LC system. However, it was not necessary to precipitate out the protein in the CE method used as it involved injecting the incubation mixture directly onto the capillary. The reaction is stopped by the separation of the individual components in the capillary, and being separated, they are no longer in contact with each other and able to react. 7-Hydroxycoumarin is seen after 0.9 min (Fig. 3) using the CE method but it takes up to 12 min using the LC method of Egan and O’Kennedy [20]. Furthermore, up to 500 injections were made onto the CE capillary during these studies without any visible degradation of capillary performance. If,
105
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4.5 -
0 z z
4.0-
:
3.5-
g 0 E
3.0-
9 z 'C ;
2.5 2.0-
: z
1.5-
:: Gi $
L
1.0 -
0.5 -
O.o;.,. 0
20
I.,.,. 40 60
60
I',. 100
120
0
Time (minutes) Fig. 5. Plot of 7-hydroxycoumarin (mnol/ml/mg protein) rt S.D. versus time for microsome preparation P17 metabolism study by LCandCE.
however, one compares the sensitivity of the available LC system 1201 with the sensitivity of the CE system developed there is no difference observed. 3.3. NADP ‘/ NmPH
c
I
,
20
,
,
40
.
,
60
.
,
80
I
,
100
.
,
c
120
Time (minutes) Fig. 4. Plot of concentration of 7-hydroxycoumarin (omol/ml/mg protein) f S.D. versus time for five microsome preparations.
ratio study
As part of the study we discovered that it was possible to monitor an integral part of the monooxygenase reaction, i.e., the NADPH-dependent coumarin metabolism reaction. One could follow the effect on both the NADP+ and the NADPH present as a result of the NADPH regeneration system in the incubation mixture (Eq. 1). To slow down the production of the NADPH from NADP+, the concentration of the glucose-6-phosphate dehydrogenase was reduced to 0.1 U/ml as compared to 4 U/ml for the metabolism study. Fig. 6 shows a plot of NADP+/NADPH absorbance ratio versus time for the incubation mixture and also for incubations in the absence of coumarin, or microsomes. The NADP+/NADPH ratio was calculated from ab-
B. Deasy et al./Anulytica ChimicaActa 310 (lW5) 101-107
106
q
A 0
7.0
14.0
No coumatln present Cormarin present (24 No microsomes present
21.0
Time
23.0
n&11)
reactions is very promising since the injection of the neat mixture onto the capillary allows results to be obtained in near real time, possibly allowing the automation of the sample transfer and measurement. The coumarin metabolism profiles determined by this capillary electrophoresis method showed that there is inter-individual variability in the metabolism of this drug, and this was in good agreement with other work in this area. To determine 7-hydroxycoumarin at lower concentrations, laser-induced fluorescence detection could be used. The metabolism of coumarin is well documented in vitro at a microsoma1 level and in vivo for urine, plasma and serum levels in humans. The method was also shown to be very useful in the determination of the metabolic cofactors NADPH and NADP+.
35.0
(minutes)
Fig. 6. Plot of NADP+/NADPH absorbance ratio versus time, for each of the controls and the incubation mixture. The controls included the incubation mixture without coumarin or without microsomes.
sorbances at 210 nm. Comparing the controls to the incubation complete incubation mixture, the NADP+/NADPH absorbance ratio changes as the cytochrome P450 utilises the NADPH for the metabolism of the coumarin. However, other biological processes in the microsomes also utilise the NADPH in the controls. Thus, the CE method described in this paper, besides analysing the metabolites can also monitor the cofactors necessary for the metabolism and this can be used to monitor whether drugs are metabolised using an NADPH system or not.
4. Conclusions The use of capillary electrophoresis in the study of the metabolism of coumarin by human liver microsomes was found to be straightforward and reliable and, in comparison with more established methods (e.g., LC), was much more rapid to perform and involved no sample preparation. The use of this analytical tool in the study of cytochrome P450
Acknowledgements We would like to thank Schaper and Brummer, Germany, the Coumarin Research Fund, The Research and Postgraduate Studies Committee, Dublin City University and Forbairt for their financial support. We would also like to thank Mr. Aidan Smyth, of Beckman Instruments Ltd., for his invaluable help.
References [l] T.M. Devlin (Ed.), Textbook of Biochemistry, 2nd edn., Wiley, New York, Chap. 23, p. 982. [2] U. Fuhr, T. Wolff, S. Harder, P. Schymanski and A.H. Staib, Drug Metabolism and Disposition, 18 (1990) 1005. [3] U. Fuhr, K. Klittich and AH. Staib, Br. J. Clin. Pharmacol., 35 (1993) 431. [4] U. Fuhr, G. Strobl, F. Manaut, E.M. Anders, F. Sorgel, E.L.D. Brinas, D.T.W. Chu, A.G. Pemet, G. Mahr, F. Sanz and A.H. Staib, Molecular Pharmacology, 43 (1993) 191. [s] U. Fuhr, E.M. Anders, G. Mahr, F. Sorgel and A.H. Staib, Antimicrobial Agents and Chemotherapy, 36 (1992) 942. [6] D. Egan, R. O’Kennedy, E. Moran, D. Cox, E. Prosser and R.D. Thomes, Drug Metab. Rev., 22 (1990) 503. [7] J. Casley-Smith, J. Ir. Coll. Phys. Surg., 22 (1993) 67. [8] 0. Pelkonen, H. Raunio, A. Rautio, J. Maenpaa and M. Lang, J. Ir. Coll. Phys. Surg., 22 (1993) 24. [9] S. Sharifi, E. Lotterer, H.C. Michaelis and J. B&her, J. Ir. Coll. Phys. Surg., 22 (1993) 29. [lo] M.E. Marshall, J.L. Mohler, K. Edmonds, B. Williams, M. Ryles, L. Weiss, D. Urban, A. Bueschen, M. Markiewicz and
B. Deasy et al. /Analytica Chimica Acta 310 (1995) 101-107 G. Cloud, J. Cancer Res. Clin. Oncol., 120 (suppl.) (1994) s39. [ll] N.W. Smith and M.B. Evans, J. Pharm. Biomed. Anal., 12 (1994) 579. [12] P. Morin and M. Dreux, J. Liq. Chromatogr., 16 (1993) 3734. [13] S. Busch, J.C. Kraak and H. Poppe, J. Chromatogr., 635 (1993) 119. 1141 P. Gariel, J.P. Gramond and F. Guyon, J. Chromatogr., 615 (1993) 317. [15] K.J. Potter, R.J.B. Allington and J. Algaier, J. Chromatogr., 652 (1993) 427.
107
[16] D.P. Bogart, B. Deasy, R. O’Kennedy, M.R. Smyth and U. Fuhr, J. Chromatogr., 663 (1994) 371. [17] J.H. Fentem, M.J. Garle and J.R. Fry, Human Exp. Toxic& 9 (1990) 329. [18] S. Sharifi, H.C. Michaelis, E. Letterer and J. Bircher, J. Liq. Chromatogr., 16 (1993) 1263. [19] T. Wolff and S. Hesse, B&hem. Pharmacol., 26 (1977) 783. [20] D. Egan and R. O’Kennedy, J. Chromatogr., 582 (1993) 137. [21] 0. Pelkonen, The 1993 Oswald Schmiedeberg Lecture, University of Oulu, Finland, p. 13.
ACTA Analytica &mica Acta 310 (1995) 101-107
Study of coumarin metabolism by human liver microsomes using capillary electrophoresis Brian Deasy a, Declan P. Bogan b, Malcolm R. Smyth a**, Richard O’Kennedy b, Uwe Fuhr ’ a School of Chemical Sciences, Dublin City University, Dublin 9, Ireland b School of Biological Sciences, Dublin City University, Dublin 9, Ireland ’ Department of Clinical Pharmacology, University Hospital, D-60590 Frankfurt am Main, Germany Received 17 January 1995; revised 8 February 1995; accepted21 February 1995
Abstract A method for the rapid determination of 7-hydroxycoumarin, based on separation by capillary electrophoresis (CE), with UV absorbance detection at 210 nm, was applied to a study of the metabolism of coumarin in human liver microsomes. The linear detection range for 7-hydroxycoumarin was O-SO pg/ml, while the limit of detection was 1 pg/ml. Human liver microsomes from 5 patients were used in metabolic studies and the profiles clearly indicated that there is inter-individual variability in coumarin 7-hydroxylase activity. The CE method allowed the investigation of NADP+/NADPH cofactor reactions involved in coumarin metabolism.The CE method was also compared to a liquid chromatographic method for the analysis of the metabolite and it was found that the results did not differ but the CE method was more rapid and needed no sample preparation. The very short time between sampling and analysis makes the method suitable for automated sample transfer and measurement. Keywords: Electrophoresis; Coumarins; Liver microsomes
1. Introduction The metabolism of coumarin (SH) in the human body can be described by reaction 1 (Fig. 1) NADPH+H++02+SH P450 + NADP+
+ H,O + SOH
(1)
where P450 is a cytochrome P450 enzyme and SOH is 7-hydroxycoumarin. NADPH (nicotinamide
* Corresponding author.
adenine dinucleotide phosphate) is needed to donate 2 electrons to the heme iron of cytochrome P450 [l]. Microsomal preparations from human liver contain cytochrome P450 isoforms and have been used to study the metabolic pathways of drugs [2-51. NADPH is regenerated by the use of glucosed-phosphate dehydrogenase and is thus recycled. Coumarin, a naturally occurring plant constituent, has been used in the treatment of cancer [6] and oedemas [7]. After administration, in humans, coumarin is quickly metabolised to 7-hydroxycoumarin by a specific cytochrome P450 (P45OCoh) in the liver [8] and therefore may be used to monitor
0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved 0003-2670(95)00136-O
SSDI
102
B. Deasy et al./Analytica
cty 0
COUMARIN
p450,
Chimica Acta 310 (1995) 101-107
HoyJy 'I-HYDROXYCOUMARlN
Fig. 1. Conversion of coumarin to 7-hydroxycoumarin.
NADPH and NADP+ migration. The electrolyte was prepared daily by mixing 0.02 M K,HPO, and 0.005 M KH,PO, in deionised water. Coumarin (2.05 X 10m4 M in 0.1% methanol: 99.9% ultra pure water) was donated by Schaper and Brummer. 2.2. Tissue preparation
activity of the isoform. It has also been suggested that 7-hydroxycoumarin is a prodrug for coumarin [9] and it is currently being investigated in clinical trials for its effectiveness in cancer treatment [lo]. Capillary electrophoresis (CE) is a recent addition to the tools available in the analytical and research laboratory, and its range of applications is dramatically increasing, e.g., for the analysis of drugs, and biological macromolecules [ll-151. The metabolism of coumarin has been monitored in the past by liquid chromatography (LC) [17,18]. However, one needs to remove the protein in the sample to protect the LC system from injection to detection. In a recent paper we have described a CE method for the determination of 7-hydroxycoumarin in urine and serum [16]. This new paper describes the application of this method to follow the metabolism of coumarin directly in the presence of liver microsomal preparations without any sample preparation. Furthermore, the analysis time for the metabolite is considerably reduced in comparison to LC methods [17,18]. The method applied also allowed the monitoring of NADPH and NADP+ levels present, and to assess their utilisation in the metabolic system.
2. Experimental 2.1. Chemicals 7-Hydroxycoumarin, NADPH, NADP+, D-glucose-6-phosphate and glucosed-phosphate dehydrogenase were purchased from Sigma (St. Louis, MO). KH a PO, and K, HPO, were obtained from Kiedel-de Haen (Hannover). 7-Hydroxycoumarin standards were prepared from a 1 mg/ml stock solution in methanol (HPLC grade, Labscan, Dublin): deionised water (10:90, v/v> by dilution in deionised water. The electrolyte solution used when monitoring metabolite formation was 0.025 M phosphate buffer, pH 7.0. The pH was adjusted to 7.5 when monitoring
The five donors of human microsomes were patients either with carcinoma of the colon or small intestine and a single liver metastasis, or patients with primary liver tumours, who all underwent partial hepatectomy. Portions of macroscopically healthy liver, taken from the excised lobe, were chilled immediately in 1.15% KC1 (0” C) and were then frozen in liquid nitrogen within a few minutes. Microsomes were obtained by differential centrifugation using the sucrose method [19]. The characteristics of the donors are summarised in Table 1. The protein concentrations of these microsome preparations were calculated using the bicinchoninic acid (BCA) assay (Pierce).
2.3. Incubation solution The concentrations of the NADPH-generating system in all incubations were 4 U/ml glucosed-phosphate dehydrogenase, 2 mg/ml p-glucosed-phosphate, and 1.2 mM NADP+. This was higher than that required to maintain maximum metabolic rates under the chosen in vitro conditions. The coumarin (substrate) concentration in the incubation solutions was always 1.6 X low4 M. For the incubations 195 ~1 of a coumarin standard (30 pg/ml, prepared in phosphate buffer, pH 7.5, 25 mM>, 10 ~1 of D-glucose-6-phosphate (50 mg/ml), 15 ~1 of NADP+ (20 mM) and 5 ~1
Table 1 Details of liver donors Liver
Donor sex
Donor age (years)
Smoking status
P13 P14 P17 P18 Rl
F F F F M
64 48 54 44 54
NS NS S NS NS
B. Deasy et al./Analytica Chimica Acta 310 (1995) 101-107
glucose-6-phosphate dehydrogenase were mixed together in an Eppendorf tube to give final concentrations as above. The reaction was started by the addition of 25 ~1 of microsomal suspension (the microsomal preparations are called P13, P14, P17, P18 and Rl), resulting in a final protein concentration of about 2 mg/ml. Following mixing, the open Eppendorf tube was transferred immediately into the incubation bath (37” Cl, and a 20 ~1 sample was taken and analysed. The metabolite formation was monitored over a 2 h period, taking samples at 0, 7, 14, 20, 30, 45, 60, 90 and 120 min, respectively. These samples were analysed using a Beckman capillary electrophoresis instrument (P/ACE System 2050). In the case of analysis by LC, the incubation was first stopped by the addition of 50 ~1 of ice-cold trichloroacetic acid (20% w/v> and centrifuged (low speed) before injection. The LC analysis was carried out according to Egan and O’Kennedy [20]. 2.4. NADP ‘/NADPH
ratio study
103
mal preparations, the concentration being calculated from a plot of absorbance versus standard concentration ( pg/ml). The linear detection range for 7-hydroxycoumarin was O-50 pg/ml and the limit of detection was 1 pg/ml.
3. Results and Discussion
3.1. Development of the CE separation The CE method developed for monitoring 7-hydroxycoumarin in plasma and urine [15] was adapted for studying the metabolism of coumarin by human liver microsomes. When interested in NADP+ (2 mM) and NADPH, a pH of 7.5 was required as these compounds exhibit a very poor peak shape in pH 7.0 electrolyte (phosphate buffer, 25 mM). The absorbance of coumarin and 7-hydroxycoumarin could both be monitored with good sensitivity at 210 nm. Fig. 2 shows an overlay of the typical electropherograms at 0 min and after 45 min for the metabolism
For this study, the concentration of glucosedphosphate dehydrogenase was reduced to 0.1 U/ml. The remainder of the incubation mixture was as above. The study was carried out in the presence and in the absence of coumarin, or the microsomal preparation. 2.5. Capillary electrophoresis separation The capillary used was a 27 cm X 50 pm fused silica column (Beckman), with a capillary to detector distance of 19.3 cm. The preparation step for priming of the capillary was a 1 min rinse with 0.1 M sodium hydroxide, followed by a 1 min rinse with electrolyte solution (0.025 M phosphate buffer, pH 7.0 or pH 7.5, depending on the separation required). The sample was applied to the capillary by a 3 s pressurised injection (0.5 p.s.i.1 and separation achieved with an applied voltage of 20 kV (rise time 0.2 min) at 25” C. Typical running current was 100 PA. The resultant separation was monitored at 210 nm with a fixed wavelength detector using Beckman System Gold” software. Concentrations of 7-hydroxycoumarin were calculated from the mean of three standard curves prepared by spiking standards of 7-hydroxycoumarin into three different microso-
0.03
,E s Lu
a 0.02 (D
co B
d
2
0.01
0.00 0.0
0.6
1.0
1.6
2.0
2.6
9.0
2.5
Time (minutes) Fig. 2. Overlay of electropherograms showing separation of (A) all neutral compounds including coumarin, (B) 7-hydroxycoumarin, (C) microsomes, (D) NADP+ and (El NADPH, at 0 min (solid line) and at 45 min (dashed line) monitored at 210 nm using 25 mM phosphate buffer at pH 7.5.
104
B. Deasy et al./Analytica
Chimica Acta 310 (1995) 101-107
0.016
jl
c
E z Z 8 5
0.011
?2 % 9
J 0.006
I
0.6
I 1.0
Time
I
c
I
1.2
1.4
(mlnutes)
Fig. 3. Overlay of electropherograms showing separation of (A) all neutral compounds including coumsrin, (B) 7-hydroxycoumarin, (C) microsomes, at 0 min (solid line) and at 45 min (dashed line) monitored at 210 nm using 25 mM phosphate buffer at pH 7.0.
it is possible to say that all the compounds that have longer migration times than coumarin are negatively charged and hence migrate more slowly than coumarin. 7-Hydroxycoumarin is more negatively charged than coumarin due to the hydroxyl group at the 7-position. This hydroxyl group is partially ionised at this PH. The microsomes migrate more slowly than the 7-hydroxycoumarin and are seen as a broad peak due to the different types of proteins in the microsomal suspensions. NADP+ has a structure with four negative charges and a positively charged nitrogen associated with it. This suggests an overall charge of 3 negative charges. This high overall negative charge explains the slow migration of NADP+. The NADPH molecule has a similar structure to NADP+ but the positive charge on the nitrogen is no longer present. As a result, this more negatively charged compound has the slowest migration rate of all the compounds. Control incubations included an absence of (A) coumarin, (B) microsomes, and (C) the NADPH regeneration system (Fig. 3). Coumarin was not quantifiable under these conditions as other unknown reactions were occurring producing neutral compounds which co-migrate with coumarin. The neutral compound produced was dependent on the NADPH reaction as observed with control (A). 3.2. Coumarin metabolism profiles
incubation for the P18 microsomal preparation. The metabolite, 7-hydroxycoumarin, migrates at 0.9 minutes and its increase can be followed under the conditions outlined under Experimental above. However, it is not well separated from the broad peak of the microsomal preparations at pH 7.5 and thus it must be monitored at pH 7.0 (Fig. 31 to resolve the compounds. It can be seen from Fig. 2 that NADP+ and NADPH are well separated and quantifiable at this pH. The use of a micellar-based electrolyte solution was also assessed, but there was no benefit found in utilising such a system for the determination of 7-hydroxycoumarin. A neutral marker (benzamide) was run under the conditions above, and it was found to co-migrate with coumarin, thus suggesting that coumarin is behaving as a neutral molecule at this pH. This is not surprising when the structure is considered (Fig. 1). By knowing the migration time of the neutral marker
Microsome preparations from 5 different patients were used in metabolism studies. Incubations were carried out in triplicate. Fig. 4 shows a plot of concentration of 7-hydroxycoumarin (nmol/ml/mg protein f SD.), versus time for each of the five metabolic studies. It shows that each of the microsomal preparations has a different coumarin metabolism profile; this is in agreement with results from other groups [ 171. In fact, the inter-individual variability of human coumarin 7-hydroxylase activity in liver microsomes is very large, and can be up to 100 fold [21]. Although the errors in concentration calculations are relatively high, the results correspond to microsomal drug metabolism results from other researchers [2]. Fig. 5 shows the comparison of using LC against CE for the analysis of the metabolite formation. It can be seen that there is no significant difference between the results from both methods.
B. Deasy et al./Analytica Chimica Acta 310 (1995) 101-107
LC is the common method used for this analysis of metabolites in cytochrome P450 studies 12-51 and also for the quantification of 7-hydroxycoumarin. The LC methods involve stopping the enzyme reactions by precipitating out the protein to minimise degradation of the LC system. However, it was not necessary to precipitate out the protein in the CE method used as it involved injecting the incubation mixture directly onto the capillary. The reaction is stopped by the separation of the individual components in the capillary, and being separated, they are no longer in contact with each other and able to react. 7-Hydroxycoumarin is seen after 0.9 min (Fig. 3) using the CE method but it takes up to 12 min using the LC method of Egan and O’Kennedy [20]. Furthermore, up to 500 injections were made onto the CE capillary during these studies without any visible degradation of capillary performance. If,
105
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Time (minutes) Fig. 5. Plot of 7-hydroxycoumarin (mnol/ml/mg protein) rt S.D. versus time for microsome preparation P17 metabolism study by LCandCE.
however, one compares the sensitivity of the available LC system 1201 with the sensitivity of the CE system developed there is no difference observed. 3.3. NADP ‘/ NmPH
c
I
,
20
,
,
40
.
,
60
.
,
80
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,
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.
,
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Time (minutes) Fig. 4. Plot of concentration of 7-hydroxycoumarin (omol/ml/mg protein) f S.D. versus time for five microsome preparations.
ratio study
As part of the study we discovered that it was possible to monitor an integral part of the monooxygenase reaction, i.e., the NADPH-dependent coumarin metabolism reaction. One could follow the effect on both the NADP+ and the NADPH present as a result of the NADPH regeneration system in the incubation mixture (Eq. 1). To slow down the production of the NADPH from NADP+, the concentration of the glucose-6-phosphate dehydrogenase was reduced to 0.1 U/ml as compared to 4 U/ml for the metabolism study. Fig. 6 shows a plot of NADP+/NADPH absorbance ratio versus time for the incubation mixture and also for incubations in the absence of coumarin, or microsomes. The NADP+/NADPH ratio was calculated from ab-
B. Deasy et al./Anulytica ChimicaActa 310 (lW5) 101-107
106
q
A 0
7.0
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No coumatln present Cormarin present (24 No microsomes present
21.0
Time
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reactions is very promising since the injection of the neat mixture onto the capillary allows results to be obtained in near real time, possibly allowing the automation of the sample transfer and measurement. The coumarin metabolism profiles determined by this capillary electrophoresis method showed that there is inter-individual variability in the metabolism of this drug, and this was in good agreement with other work in this area. To determine 7-hydroxycoumarin at lower concentrations, laser-induced fluorescence detection could be used. The metabolism of coumarin is well documented in vitro at a microsoma1 level and in vivo for urine, plasma and serum levels in humans. The method was also shown to be very useful in the determination of the metabolic cofactors NADPH and NADP+.
35.0
(minutes)
Fig. 6. Plot of NADP+/NADPH absorbance ratio versus time, for each of the controls and the incubation mixture. The controls included the incubation mixture without coumarin or without microsomes.
sorbances at 210 nm. Comparing the controls to the incubation complete incubation mixture, the NADP+/NADPH absorbance ratio changes as the cytochrome P450 utilises the NADPH for the metabolism of the coumarin. However, other biological processes in the microsomes also utilise the NADPH in the controls. Thus, the CE method described in this paper, besides analysing the metabolites can also monitor the cofactors necessary for the metabolism and this can be used to monitor whether drugs are metabolised using an NADPH system or not.
4. Conclusions The use of capillary electrophoresis in the study of the metabolism of coumarin by human liver microsomes was found to be straightforward and reliable and, in comparison with more established methods (e.g., LC), was much more rapid to perform and involved no sample preparation. The use of this analytical tool in the study of cytochrome P450
Acknowledgements We would like to thank Schaper and Brummer, Germany, the Coumarin Research Fund, The Research and Postgraduate Studies Committee, Dublin City University and Forbairt for their financial support. We would also like to thank Mr. Aidan Smyth, of Beckman Instruments Ltd., for his invaluable help.
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B. Deasy et al. /Analytica Chimica Acta 310 (1995) 101-107 G. Cloud, J. Cancer Res. Clin. Oncol., 120 (suppl.) (1994) s39. [ll] N.W. Smith and M.B. Evans, J. Pharm. Biomed. Anal., 12 (1994) 579. [12] P. Morin and M. Dreux, J. Liq. Chromatogr., 16 (1993) 3734. [13] S. Busch, J.C. Kraak and H. Poppe, J. Chromatogr., 635 (1993) 119. 1141 P. Gariel, J.P. Gramond and F. Guyon, J. Chromatogr., 615 (1993) 317. [15] K.J. Potter, R.J.B. Allington and J. Algaier, J. Chromatogr., 652 (1993) 427.
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[16] D.P. Bogart, B. Deasy, R. O’Kennedy, M.R. Smyth and U. Fuhr, J. Chromatogr., 663 (1994) 371. [17] J.H. Fentem, M.J. Garle and J.R. Fry, Human Exp. Toxic& 9 (1990) 329. [18] S. Sharifi, H.C. Michaelis, E. Letterer and J. Bircher, J. Liq. Chromatogr., 16 (1993) 1263. [19] T. Wolff and S. Hesse, B&hem. Pharmacol., 26 (1977) 783. [20] D. Egan and R. O’Kennedy, J. Chromatogr., 582 (1993) 137. [21] 0. Pelkonen, The 1993 Oswald Schmiedeberg Lecture, University of Oulu, Finland, p. 13.