Metabolism of methylisoeugenol in liver microsomes of human, rat, and bovine origin

Article abstract of DOI:10.1124/dmd.111.038851

Methylisoeugenol (1,2-dimethoxy-4-propenylbenzene, 1) is a minor constituent of essential oils, naturally occurring as a mixture of cis/trans isomers. 1 is a U.S. Food and Drug Administration-approved food additive and has been given "Generally Recognized as Safe" status. Previously, metabolism of 1 has been studied in the rat, revealing mainly nontoxic cinnamoyl derivatives as major metabolites. However, data concerning the possible formation of reactive intermediary metabolites are not available to date. In this study, the oxidative metabolism of 1 was studied using liver microsomes of rat [not induced, rat liver microsomes (RLM); Aroclor1254 induced RLM (ARLM)], bovine, and human (pooled from 150 donors) origin. Incubations of these microsomes with 1 provided phase I metabolites that were separated by high-performance liquid chromatography (HPLC) and identified by NMR and UV-visible spectroscopy and/or liquid chromatography-mass spectrometry. Identity was confirmed by comparison with ¹H NMR spectra of synthesized reference compounds. Formation of metabolites was quantified by HPLC/UV using dihydromethyleugenol (10) synthesized as the internal standard. From incubations of ARLM with 1, seven metabolites could be detected, with 3′- hydroxymethylisoeugenol (2), isoeugenol and isochavibetol (3 + 4), and 6-hydroxymethylisoeugenol (5) being the main metabolites. Secondary metabolites derived from 1 were identified as the α,β-unsaturated aldehyde 3′-oxomethylisoeugenol (6) and 1′,2′-dihydroxy- dihydromethylisoeugenol (7). We were surprised to find that formation of allylic 6-hydroxymethyleugenol (8) was observed starting at approximately 30 min after the beginning of incubations with ARLM. HLM did not form ring-hydroxylated metabolites but were most active in the formation of 6 and 7. ARLM incubations displayed the highest turnover rate and broadest metabolic pattern, presumably resulting from an increased expression of cytochrome P450 enzymes. In conclusion, we present a virtually complete pattern of nonconjugated microsomal metabolites of 1 comprising reactive metabolites and suggest the formation of reactive intermediates that need more investigation with respect to their possible adverse properties. Copyright

Full text of DOI:10.1124/dmd.111.038851

0090-9556/11/3909-1727–1733$25.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
DMD 39:1727–1733, 2011
Vol. 39, No. 9
38851/3707838
Printed in U.S.A.
Metabolism of Methylisoeugenol in Liver Microsomes of Human,
Rat, and Bovine Origin
Alexander T. Cartus, Karl-Heinz Merz, and Dieter Schrenk
Food Chemistry and Toxicology, University of Kaiserslautern, Kaiserslautern, Germany
Received February 17, 2011; accepted June 1, 2011
ABSTRACT:
Methylisoeugenol (1,2-dimethoxy-4-propenylbenzene, 1) is a minor (10) synthesized as the internal standard. From incubations of
constituent of essential oils, naturally occurring as a mixture of ARLM with 1, seven metabolites could be detected, with 3؅-hy-
cis/trans isomers. 1 is a U.S. Food and Drug Administration-ap- droxymethylisoeugenol (2), isoeugenol and isochavibetol (3 ؉ 4),
proved food additive and has been given “Generally Recognized as and 6-hydroxymethylisoeugenol (5) being the main metabolites.
Safe” status. Previously, metabolism of 1 has been studied in the
Secondary metabolites derived from 1 were identified as the ␣,␤-
rat, revealing mainly nontoxic cinnamoyl derivatives as major me- unsaturated aldehyde 3؅-oxomethylisoeugenol (6) and 1؅,2؅-dihy-
tabolites. However, data concerning the possible formation of re- droxy-dihydromethylisoeugenol (7). We were surprised to find that
active intermediary metabolites are not available to date. In this formation of allylic 6-hydroxymethyleugenol (8) was observed
study, the oxidative metabolism of 1 was studied using liver mi- starting at approximately 30 min after the beginning of incubations
crosomes of rat [not induced, rat liver microsomes (RLM); Aro- with ARLM. HLM did not form ring-hydroxylated metabolites but
clor1254 induced RLM (ARLM)], bovine, and human (pooled from were most active in the formation of 6 and 7. ARLM incubations
150 donors) origin. Incubations of these microsomes with 1 pro- displayed the highest turnover rate and broadest metabolic pat-
vided phase I metabolites that were separated by high-perfor- tern, presumably resulting from an increased expression of cyto-
mance liquid chromatography (HPLC) and identified by NMR and chrome P450 enzymes. In conclusion, we present a virtually com-
UV-visible spectroscopy and/or liquid chromatography-mass plete pattern of nonconjugated microsomal metabolites of 1
spectrometry. Identity was confirmed by comparison with 1H NMR comprising reactive metabolites and suggest the formation of re-
spectra of synthesized reference compounds. Formation of me- active intermediates that need more investigation with respect to
tabolites was quantified by HPLC/UV using dihydromethyleugenol their possible adverse properties.
Introduction
has been claimed to be nongenotoxic (Hasheminejad and Caldwell,
1994), although definite data were missing.
Methylisoeugenol (1,2-dimethoxy-4-propenylbenzene, 1; see Fig.
5) is an alkenylbenzene naturally occurring as a mixture of cis/trans
isomers in essential oils of Asarum arifolium, Cymbopogon javanen-
sis, and in nearly 60 other essential oils (Opdyke, 1975). It is used in
perfumery as an ingredient having a clove-carnation odor and is
applied as an additive to foods in the parts-per-million range.
1 was classified as ”Generally Recognized as Safe” by the Flavor
and Extract Manufacture Association in 1965 and is approved by the
U.S. Food and Drug Administration (2010) for food use. The acute
oral LD50 in rats was reported to be 1.5 and 2.5 g/kg b.wt. (Opdyke,
1975). Adverse effects have not been seen in a feeding study with rats
receiving 300 mg/kg b.wt. per day (Purchase et al., 1992). Overall, 1
When applied at full strength, 1 was irritating to skin (rabbit), but
concentrations up to 8% in petrolatum did not produce irritations on
human subjects. When an equal concentration was used, 1 did not
cause sensitization in a maximization test on human volunteers (Op-
dyke, 1975). However, sensitization was positive in a local lymph
node assay on mice (Research Institute for Fragrance Materials,
www.rifm.org).
The major metabolic pathway of 1 in the rat was found to be the
oxidative transformation of the propenyl side chain leading to hy-
droxy- and/or methoxy-substituted cinnamic and benzoic acids. Other
reactions were O-demethylation and oxidation to 3,4-dimethoxyphe-
nylacetic acid and 4-hydroxy-3-methoxyphenylacetone. Epoxidation
of the side chain appeared to be a minor metabolic reaction (Solheim
and Scheline, 1976). The metabolites reported in this previous study
suggest the formation of reactive intermediates that may lead to
hapten formation or may trigger other adverse effects. In our study,
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.111.038851.
ABBREVIATIONS: 1, methylisoeugenol, 2, 3Ј-hydroxymethyleugenol; 3, isoeugenol; 4, isochavibetol; 5, (E)-6-hydroxymethylisoeugenol; 6,
(E)-3Ј-oxomethylisoeugenol; 7, 1Ј,2Ј-dihydroxymethylisoeugenol; 8, 6-hydroxymethyleugenol; 9, 1Ј-hydroxymethyleugenol; 10, dihydromethyl-
eugenol; 11, methyleugenol; 12, methylisoeugenol-1,2-epoxide; 13, 2Ј3Ј-dihydroxymethyleugenol; 14, (E)-3,4-dimethoxycinnamic acid; HPLC,
high-performance liquid chromatography; IR, infrared; ARLM, Aroclor1254 pretreatment rat liver microsomes; BLM, bovine liver microsomes;
P450, cytochrome P450; DAD, diode array detector; HLM, human liver microsomes; RLM, rat liver microsomes; DMSO, dimethyl sulfoxide; EtOAc,
ethyl acetate; Vis, visible.
1727

1728
CARTUS ET AL.
ities (Struktur- und Genehmigungsbeho¨rde Rheinland-Pfalz). Male Wistar rats
were purchased from Charles River (Sulzbach, Germany). Animals were kept
under a 12-h light/dark cycle and received water and commercial laboratory
chow ad libitum. For inducing experiments, rats (b.wt. 180–250 g) were given
a single dose of 500 mg/kg b.wt. Aroclor1254 i.p. from a stock solution of 200
mg/ml Aroclor1254 dissolved in corn oil. Five days after treatment the animals
were sacrificed. Aroclor1254-treated rat (ARLM), noninduced rat (RLM), and
bovine liver microsomes (BLM) were prepared from the liver of freshly
sacrificed/slaughtered animals as described for rat liver by Melancon et al.
(1985). Bovine livers were from 1.5- to 2-year-old female German Black Pied
cattle (dairy cattle) and were obtained from a local slaughterhouse. Human
liver microsomes (HLM) were purchased from BD Bioscience (Heidelberg,
Germany) from a pool derived from 150 gender-mixed donors. Protein was
measured photometrically using the method of Bradford (1976). Total cyto-
chrome P450 (P450) contents were measured according to the method of
Omura and Sato (1962).
Incubations. Incubations of microsomes (2 mg of microsomal protein/ml)
were performed with 0.1, 0.5, or 2.5 mM substrate dissolved in DMSO (final
concentration 5%) with a NADPH-regenerating system consisting of 1 mM
NADPH, 0.5 units glucose-6-phosphate dehydrogenase, 5 mM glucose 6-phos-
phate, 3 mM magnesium chloride, and 50 mM phosphate buffer, pH 7.4.
Incubations were carried out in various volumes (from 1 to 100 ml for
preparative uses) and were gently shaken in an incubator (TH-30; Carl Roth,
Karlsruhe, Germany) at 37°C. After preincubation with substrate for 5 min at
37°C, the NADPH-generating system was added and the mixture was incu-
bated for up to 24 h. Control incubations were carried out with heat-inactivated
microsomes and with intact microsomes but without the NADPH-regenerating
system, respectively. Incubations and time-course measurements were done in
triplicate as independent experiments. The high concentration of DMSO (5%)
was applied to improve solubility of the substrate, in particular of isolated
metabolites. Although DMSO is known to act as a P450 inhibitor, concentra-
tions of metabolites in control incubations of 1 using 0.5% DMSO were found
not to be significantly different (data not shown).
we intended to investigate the complete spectrum of metabolites of 1
formed by incubations with liver microsomes of different species.
Materials and Methods
Chemicals. Acetone, CDCl3, and dimethyl sulfoxide (DMSO) were pur-
chased from Merck (Darmstadt, Germany). ␤-NADP, glucose 6-phosphate,
and glucose-6-phosphate dehydrogenase were obtained from Sigma-Aldrich/
Fluka (Taufenkirchen, Germany). Methanol was obtained in high-performance
liquid chromatography (HPLC) gradient grade from J. T. Baker (Griesheim,
Germany). Aroclor1254 was originally purchased from Monsanto (St. Louis,
MO). 1, isoeugenol (3), methyleugenol (11), and DMSO-d6 were purchased
from Sigma-Aldrich (Steinheim, Germany) at Ͼ98% purity. The purity of the
chemicals was checked by HPLC analysis and/or NMR spectroscopy. Metab-
olites of 1 were synthesized according to the procedures described under
Synthesis. The nomenclature of compounds and metabolites follows the num-
bering indicated in Fig. 5 for 1.
Analytical Methods. 1H NMR and 13C NMR spectra were recorded on a
Bruker DPX 400 (Bruker BioSpin, Rheinstetten, Germany) or Avance 600
spectrometer (Bruker) in DMSO-d6 (1H NMR, ␦ 2.49 ppm; 13C NMR, ␦ 39.5
ppm) or CDCl3 (1H NMR, ␦ 7.26 ppm; 13C NMR, ␦ 77 ppm) at 298 K.
Thin-layer chromatography was performed on silica gel 60 F-254 plates
(Merck, Darmstadt, Germany). Infrared (IR) spectra were recorded on a
JASCO FT/IR-6100 spectrophotometer (Jasco, Gross-Umstadt, Germany). The
liquid chromatography-tandem mass spectrometry system consisted of a
PerkinElmer liquid chromatography system coupled to an API 2000 Q trap
instrument (Applied Biosystems, Foster City, CA). The mass spectrometer was
operated using an atmospheric pressure chemical ionization source in the
positive ion mode. Acquisition and data processing were done with Analyst
software, version 1.4 (Applied Biosystems). The instrumental settings used for
the liquid chromatography/tandem mass spectrometry analysis were as fol-
lows: ion source temperature 490°C; declustering potential 19 V; needle
current 5 V; focusing potential 364 V; entrance potential 8.5 V; curtain gas 19;
ion source gas 48; and turbo gas 52 (the latter three are given in arbitrary units
from Analyst software). Analytical HPLC was carried out using an Agilent
1100 HPLC system equipped with an autosampler (series G1329A), a quadru-
ple pumping system (series G1311A), a diode array spectrophotometer (DAD;
series G1315A), and a column heater (series G1316A) (Agilent, Waldbronn,
Germany). HPLC and liquid chromatography-tandem mass spectrometry sep-
arations were performed using a C18 column [Hibar (5 ␮m, 4 ϫ 125 mm) and
LiChrosorb; Merck, Darmstadt, Germany]. The mobile phase system consisted
of buffer A (10 mM NH4OAc in water) and buffer B (pure methanol).
Separation was achieved using a 30-min linear gradient to 70% buffer B from
initial conditions of 10% buffer B following a 5-min period with constant
eluent consistency of 70% buffer B with a total analysis time of 38 min per
sample at a flow rate of 1 ml/min. The column temperature was kept constant
at 35°C. Detection wavelengths were 230, 261, and 280 nm with a reference
wavelength of 340 nm each and the detection wavelength of 340 nm with 450
nm as reference wavelength. Injection volumes were 50 ␮l each. Preparative
HPLC was performed with an Agilent 1200 series system comprising two
preparative pumps (series G1361A), an automatic fraction collector (series
G1364B), and a multiwave detector (series G1315A). Injection was achieved
over a manual valve with sample loops of 2-ml (incubations) or 10-ml
(synthesis) volume. As solid phase, RP18 (Reprosil 100, 5 ␮m, 250 ϫ 20 mm;
Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) was used.
Sample Preparation. After incubation, samples were diluted with equal
volumes of a ice-cold solution (Ϫ20°C) of acetone containing 0.1 or 0.5 mM
10 as an internal standard. Samples were cooled for 20 min on ice. The
mixtures were shaken intensely, and the precipitated protein was removed by
centrifugation at 13,000g for 5 min. For time-course measurements, the su-
pernatants were used directly for HPLC analysis. The supernatants of single
incubations were extracted five times with an equal volume of ethyl acetate
(EtOAc). The solvent of the combined organic phases was removed, and the
residue was taken up in a defined volume of methanol before further analysis.
Synthesis. 3Ј-Hydroxymethylisoeugenol (2) was prepared according to the
method of Solheim and Scheline (1976) using 1Ј-hydroxymethyleugenol (9) as
starting material. Yield: 229 mg (1.18 mmol, 42%, light yellow oil). Analysis:
Rf ϭ 0.36 (EtOAc/hexane 1:1, v/v); 1H NMR (DMSO-d6, 400 MHz): ␦ 3.72
(s, 3H, OCH3), 3.75 (s, 3H, OCH3), 4.07 (pt, 2H, H3Ј), 4.80 (t, 1H, OH, J ϭ
5.4 Hz), 6.23 (dt, 1H, H2Ј, J ϭ 16 Hz, J ϭ 5.4 Hz), 6.44 (d, 1H, H1Ј, J ϭ 16
Hz), 6.88 (ps; 2H, H5, H6), 7.03 (s, 1H, H2); tR (HPLC-UV) ϭ 16.7 min (280
nm; 500 ␮M); UV/Vis (HPLC-DAD): ␭max (absolute absorbance in mAU) ϭ
214 (780), 263 (580), 300 (220) nm; IR (neat): 3583, 3414, 2255, 2128, 1659,
1050, 1025, 1005, 825, 764, 666 cmϪ1
.
(E)-6-Hydroxymethylisoeugenol (5) was synthesized according to Shulgin
(1965) by isomerization of 6-hydroxymethyleugenol (8). Under argon atmo-
sphere, a mixture of 8 (4.5 g, 23.2 mmol) and potassium hydroxide (11.2 g;
0.20 mol) in dried ethanol (5 ml) was stirred at 140–150°C for 7 days.
Reaction control was accomplished by analytical HPLC. Additional KOH
(11.2 g; 0.20 mol) was added after 48 and 96 h, each. The reaction was stopped
after 7 days because no further conversion was observed. Five hundred
milliliters of water were added, and the mixture was acidified with dilute
sulfuric acid and extracted with ethyl acetate (four times, 150 ml each). The
combined organic layers were dried (MgSO4) and the solvent was evaporated
Quantification. For quantification of the formed microsomal metabolites,
the synthesized reference compounds were measured at concentrations of 1,
10, 50, 100, 500, and 1000 ␮M in methanol with the HPLC gradient described
under Analytical Methods. Injection volumes were 50 ␮l each. Peak areas (area
under the curve) detected at four different wavelengths (␭ ϭ 230, 261, 280, and
340 nm) were plotted versus concentration. Metabolite concentrations were
calculated with the slope of the linear regression (R2 Ͼ 0.99 for each com-
pound) at the most sensitive wavelength.
Internal Standard. As internal standard, dihydromethyleugenol (1,2-dime- in vacuo. The crude product (4.2 g) was purified by column chromatography
thoxy-4-propylbenzene, 10) was synthesized according to the method given using silica gel and ethyl acetate/hexane (1:2). Crystallization from ethyl
under Synthesis. Its retention time was 31 min, a time when no metabolites acetate/pentane yielded the pure trans-isomer 5 (2.8 g, 14.4 mmol, 62%) as
were found to elute.
light yellow needles. Analysis: Rf ϭ 0.41 (EtOAc/hexane 1:2), 1H NMR
Microsomes. All animal experiments (rats) were performed according to (DMSO-d6, 400 MHz) ␦ 1.7 (dd, 3H, CH3, J ϭ 6.4 Hz, J ϭ 1.2 Hz); 3.66 (s,
National Animal Welfare Regulations after authorization by the local author- 6H, 2 OCH3); 6.04 (m, 1H, H2Ј); 6.41 (s, 1H, H5); 6.51 (dd, 1H, H1Ј, J ϭ 16.4

METABOLISM OF METHYLISOEUGENOL IN LIVER
1729
Hz, J ϭ 1.4 Hz); 6.83 (s, 1H, H2); 9.05 (s; 1H, OH); 13C{1H}-NMR (DMSO- min (280 nm; 500 ␮M); UV/Vis (HPLC-DAD): ␭max (absolute absorbance in
mAU) ϭ 230 (260), 290 (170) nm.
d6, 25°C, 100 MHz) ␦ 18.6, 55.3; 56.2; 100.9; 110; 115.5; 121.9; 125.6; 142;
148.2; 148.6 ppm; IR (KBr): 3405, 3040, 3009, 2995, 2956, 2932, 2848, 1615,
1524, 1454, 1419, 1375, 1297, 1266, 1204, 1115, 1033, 999, 970, 940, 873,
827, 753, 684, 538, 472 cmϪ1; tR (HPLC-UV; 261 nm, 500 ␮M) ϭ 21.5 min;
UV/Vis (HPLC-DAD): ␭max (absolute absorbance in mAU) ϭ 215 (1100), 257
(700), 315 (350) nm.
9 was prepared according to the method of Borchert et al. (1973) using
veratraldehyde as starting material. Yield: 2.25 g (11.6 mmol, 96%, light
yellow oil). Analysis: Rf ϭ 0.32 (EtOAc/hexane 1:2); 1H NMR (DMSO-d6,
400 MHz) ␦ 3.71 (s, 3H, OCH3); 3.72 (s, 3H, OCH3); 4.97 (t, 1H, H3ЈZ, J ϭ
5.1 Hz); 5.03 (d, 1H, H3ЈE, J ϭ 10.2 Hz); 5.22 (dt, 1H, H1Ј, J ϭ 17 Hz); 5.41
(d, 1H, OH, J ϭ 4.4 Hz); 5.93 (m; 1H, H2Ј); 6.82 (dd, 1H, H6, J ϭ 8.5, 1.6
Hz); 6.87 (d, 1H, H5, J ϭ 8.5 Hz); 6.90 (d, 1H, H2, J ϭ 1.6 Hz); 13C{1H}-
NMR (DMSO-d6, 25°C, 150 MHz) ␦ 55.9; 56.1; 73.7; 110.7; 112.2; 113.5;
118.8; 137.4; 142.7; 148.3; 149.1 ppm; IR (neat): 3504, 3583, 3504, 3078,
3002, 2936, 2836, 1735, 1640, 1594, 1515, 1464, 1418, 1374, 1339, 1262,
1234, 1186, 1155, 1139, 1028, 992, 926, 860, 813, 785, 763, 666 cmϪ1; tR
(E)-3Ј-Oxomethylisoeugenol (6) was synthesized according to the method
of Bu¨rgi et al. (1993) using 2 as starting material. Separation by preparative
HPLC yielded 119 mg (0.62 mmol, 47%) of the pure trans-product as pale
1
yellow crystals. Analysis: Rf ϭ 0.39 (EtOAc/hexane 1:2); H NMR (DMSO-
d6, 400 MHz) ␦ 3.80 (s, 6H, 2 OCH3); 6.78 (dd, 1H, H2Ј, J ϭ 7:8 Hz, J ϭ 15.6
Hz); 7.02 (d, 1H, H5, J ϭ 8.2 Hz); 7.27 (dd, 1H, H6, J ϭ 8.2 Hz, J ϭ 1.6 Hz);
7.35 (d, 1H, H2, J ϭ 1.6 Hz); 7.62 (d, 1H, H1Ј, J ϭ 15.6 Hz); 9.60 (d, 1H, H3Ј,
J ϭ 7.8 Hz); IR (KBr): 3437, 2929, 2838, 1667, 1619, 1597, 1512, 1469, 1426,
1400, 1273, 1225, 1165, 1129,1037, 1017, 984, 794, 581 cmϪ1; tR (HPLC-
(HPLC-UV) ϭ 16.4 min (280 nm; 500 ␮M); UV/Vis (HPLC-DAD): ␭
(absolute absorbance in mAU) ϭ 233 (280), 278 (100) nm.
max
10 was prepared in a hydrogenation apparatus, 289 mg of Pd/C (10%) were
added to a solution of 11 (3 g; 16.8 mmol) in ethanol (30 ml). The suspension
was stirred vigorously under slight excess pressure at room temperature until
381 cm3 of hydrogen (17 mmol) was taken up. The charcoal was filtered off,
and the solvent was removed in vacuo. Yield: 1.09 g (6.60 mmol, 99%,
colorless oil). Analysis: Rf ϭ 0.57 (EtOAc/hexane 1:4); 1H NMR (DMSO-d6,
400 MHz) ␦ 0.86 (t, 3H, H3Ј, J ϭ 7.5 Hz); 1.54 (sext, 2H, H2Ј, J ϭ 7.5 Hz);
2.46 (t, 2H, H1Ј, J ϭ 7.5 Hz); 3.69 (s, 3H, OCH3); 3.71 (s, 3H, OCH3); 6.66
(dd, 1H, H6, J ϭ 8.2 Hz, J ϭ 1.7 Hz); 6.75 (d, 1H, H2, J ϭ 1.7 Hz); 6.81 (d,
1H, H5; J ϭ 8.2 Hz); IR (KBr): 2997, 2957, 2932, 2870, 2834, 1607, 1590,
1516, 1465, 1416, 1377, 1341, 1262, 1236, 1191, 1156, 1141, 1092, 1031, 846,
806, 764, 666 cmϪ1; tR (HPLC-UV) ϭ 31 min (280 nm; 500 ␮M); UV/Vis
UV) ϭ 19 min (261 nm; 1 mM); UV/Vis (HPLC-DAD): ␭
(absolute
max
absorbance in mAU) ϭ 230 (2600), 240 (3000), 300 (3000), 360 (2750) nm.
For synthesis of 1Ј,2Ј-dihydroxymethylisoeugenol (7), a solution of 1 (1 g,
5.61 mmol) in dichloromethane (15 ml) was added to a mixture (20 ml 1:1,
v/v) of a solution of saturated sodium bicarbonate and a solution of saturated
ammonium chloride. A solution of meta-chloroperbenzoic acid (70%, 1.45 g,
5.88 mmol) in dichloromethane (10 ml) was added dropwise. The mixture was
vigorously stirred for 2 h at room temperature. The organic layer was sepa-
rated, and the aqueous layer was extracted with dichloromethane three times.
The combined organic phases were washed three times with water, dried over
MgSO4, and the solvent was removed in vacuo to yield 1-(3,4-dimethoxyphe-
nyl)-1-hydroxypropane-2-yl-3-chlorobenzoate (1.90 g 5.42 mmol; 97%) as a
white solid. Analysis: Rf ϭ 0,31 (EtOAc/hexane 1:2); 1H NMR (DMSO-d6,
400 MHz): ␦ 0.95 (d, 3H, CH3); 3.71 (s, 3H, OCH3); 3.74 (s, 3H, OCH3); 4.05
(sext, 1H, H2Ј); 5,14 (d, 1H, H1Ј); 5.59 (d, 1H, OH); 6.90 (d, 1H, H5, J ϭ 7.8
Hz); 6.94 (dd, 1H, H6, J ϭ 7.8 Hz, J ϭ 1.7 Hz); 7 (d, 1H, H2, J ϭ 1.7 Hz);
7.56 (t, 1H, H5Љ, J ϭ 7.8 Hz); 7.72 (dd, 1H, H4Љ, J ϭ 8.2 Hz, J ϭ 1.4 Hz); 7.98
(d, 1H, H6Љ, J ϭ 7.8 Hz); 8.07 (t, 1H, H2Љ, J ϭ 1.4 Hz); IR (KBr): 3515, 3069,
2972, 2936, 2908, 2837, 1721, 1606, 1594, 1518, 1465, 1422, 1346, 1321,
1256, 1161, 1139, 1087, 1073, 1027, 978, 897, 866, 849, 809, 793, 749, 737,
(HPLC-DAD): ␭
(absolute absorbance in mAU) ϭ 230 (270), 280
max
(100) nm.
2Ј,3Ј-Dihydroxymethyleugenol (13) was synthesized with 2Ј,3Ј-epoxy-
methyleugenol (101 mg; 0.52 mmol, prepared by the method of Fieser, 1967).
The starting material was mixed with 5 ml of water at 25°C. Solid potassium
hydroxide (270 mg, 15.1 mmol) was added, and the mixture was refluxed for
2 h. After cooling to room temperature, 6 N HCl was added up to pH of 5 to
6. The mixture was extracted three times with ethyl acetate, and the combined
organic layers were dried (MgSO4), filtered, and concentrated in vacuo to yield
13 as a white solid (76 mg, 0.36 mmol, 69%). Analysis: Rf ϭ 0.19 (EtOAc/
hexane 1:2); 1H NMR (DMSO-d6, 600 MHz) ␦ 2.45 and 2.67 (m, 2H, H1Ј),
3.26 (m, 2H, H3Ј), 3.59 (m, 1H, H2Ј), 3.69 (s, 3H, OCH3), 3.71 (s, 3H,
OCH3), 4.51 (ps, br, 2H, OH), 6.69 (dd, 1H, H6, J ϭ 8.2 Hz, J ϭ 1.8 Hz), 6.79
(d, 1H, H2, J ϭ 1.8 Hz), 6.81 (d, 1H, H5, J ϭ 8.2 Hz); 1H NMR (CDCl3, 25°C,
400 MHz) ␦ 2.47 (m, 2H, H1Ј), 3.13 and 3.20 (m, 2H, H3Ј), 3.50 (s, 3H,
OCH3), 3.52 (s, 3H, OCH3), 3.63 (m, 1H, H2Ј), 6.46 (m, 3H, ArH); tR
702, 674, 665, 645 cmϪ1
.
This intermediate was used without further purification. To 1.83 g (5.23
mmol) of this intermediate in methanol (30 ml), sodium hydroxide (1.56 g, 39
mmol) in methanol (10 ml) was added. The solution was refluxed for 1 h until
no starting material was detectable by thin-layer chromatography. After cool-
ing to room temperature, the solution was acidified with 1 N HCl and adjusted
to pH 10 using a saturated solution of ammonium bicarbonate. The aqueous
layer was extracted three times with EtOAc (30 ml). The combined organic
layers were washed with brine and water, dried (MgSO4), and the solvent was
evaporated in vacuo. Yield: 1 g (4.74 mmol, 91%, white solid). Analysis: Rf ϭ
0.14 (EtOAc/hexane 1:3); 1H NMR (DMSO-d6, 400 MHz) ␦ 0.81 (d, 3H, H3Ј,
J ϭ 6.2 Hz); 3.61 (m, 1H, H2Ј); 4.18 (m, 1H, H1Ј); 4.51 (d, 1H, OH, J ϭ 4.3
Hz); 5.05 (d, 1H, OH, J ϭ 4.3 Hz); 6.79 (dd, 1H, H6; J ϭ 8.2 Hz, J ϭ 1.6 Hz);
6.85 (d, 1H, H5, J ϭ 8.2 Hz); 6.88 (d, 1H, H2, J ϭ 1.6 Hz); IR (KBr): 3411,
3274, 2964, 2930, 2834, 1606, 1594, 1520, 1460, 1420, 1373, 1343, 1260,
1239, 1160, 1146, 1079, 1057, 1025, 910, 819, 789, 764, 644, 618, 598, 543,
421 cmϪ1; tR (HPLC-UV) ϭ 10.8 min (280 nm; 500 ␮M); UV/Vis (HPLC-
(HPLC-UV) ϭ 10.5 min (280 nm, 1 mM); UV/Vis (HPLC-DAD): ␭
(absolute absorbance in mAU) ϭ 230 (2100), 280 (850) nm.
max
(E)-3,4-Dimethoxycinnamic acid (14) was synthesized starting from ferulic
acid (1.06 g; 5.46 mmol). After methylation (2.80 g, 20.3 mmol of K2CO3 and
2.78 g, 21.84 mmol, 2 ml of dimethyl sulfate) in acetone, the crude product was
dissolved in methanol and saponificated with KOH (2.58 g, 46 mmol) to yield
1.10 g (5.30 mmol, 97%) of pure trans-14 as a white solid. Analysis: Rf ϭ 0,24
(EtOAc/hexane 1:2); 1H NMR (DMSO-d6, 600 MHz, 295K) ␦ 3.78 (s, 3H,
OCH3); 3.79 (s, 3H, OCH3); 6.42 (d, 1H, H2Ј, J ϭ 15.8 Hz); 6.96 (d, 1H, H5,
J ϭ 8.2 Hz); 7.18 (dd, 1H, H6, J ϭ 8.2 Hz, J ϭ 2.0 Hz); 7.30 (d, 1H, H2, J ϭ 2.0
Hz); 7.50 (d, 1H, H1Ј, J ϭ 15.8 Hz); 12.19 (s, br, 1H, COOH); IR (KBr): 3447,
2937, 2841, 1683, 1625, 1596, 1584, 1516, 1457, 1426, 1408, 1340, 1315, 1299,
1263, 1210, 1169, 1141, 1024, 976, 939, 840, 811, 580 cmϪ1; tR (HPLC-UV) ϭ
6.5 min (280 nm; 0.5 mM); UV/Vis (HPLC-DAD): ␭max (absolute absorbance in
mAU) ϭ 215 (600), 230 (540), 285 (590), 312 (580) nm.
DAD): ␭
(absolute absorbance in mAU) ϭ 230 (135), 277 (35) nm.
max
8 was synthesized according to Benbow and Katoch-Rouse (2001) (Proce-
dure B) using 3,4-dimethoxyphenol (4 g, 26 mmol) as starting material.
Allylation of the phenolic hydroxyl group led to the required allylether (99%).
Subsequent Claisen rearrangement provided 8 (4.9 g, 25.2 mmol, 97%) as
white crystals. Analysis of 8: Rf ϭ 0.43 (EtOAc/hexane 1:2); 1H NMR
(DMSO-d6, 400 MHz) ␦ 3.71 (s, 3H, OCH3); 3.72 (s, 3H, OCH3); 4.97 (t, 1H,
H3ЈZ, J ϭ 5.1 Hz); 5.03 (d, 1H, H3ЈE, J ϭ 10.2 Hz); 5.22 (dt, 1H, H1Ј, J ϭ
17 Hz); 5.41 (d, 1H, OH, J ϭ 4.4 Hz); 5.93 (m; 1H, H2Ј); 6.82 (dd, 1H, H6,
J ϭ 8.5 Hz, J ϭ 1.6 Hz); 6.87 (d, 1H, H5, J ϭ 8.5 Hz); 6.90 (d, 1H, H2, J ϭ
1.6 Hz); 13C{1H}-NMR (DMSO-d6, 25°C, 100 MHz) ␦ 33.3, 55.5; 56.5; 101;
114.8; 114.9; 116.6; 137.6; 141.6; 147.8; 148.8 ppm; IR (neat): 3445, 3077,
3002, 2937, 2911, 2836, 1637, 1617, 1523, 1465, 1451, 1415, 1360, 1295,
Results
Human, bovine, and rat (Aroclor1254-induced and noninduced)
liver microsomes were incubated with 1 in the presence of a NADPH-
generating system to generate metabolites formed by metabolic phase
I reactions. Incubations without the NADPH-generating system did
not produce metabolites in detectable amounts (data not shown).
Relatively high concentrations (100 and 500 ␮M) of 1 were used with
1238, 1203, 1114, 1030, 999, 916, 847, 756, 666 cmϪ1; tR (HPLC-UV) ϭ 20 incubations to ensure detection of lesser-formed metabolites.

1730
CARTUS ET AL.
FIG. 1. Formation of metabolites and decrease of
1 during incubation of ARLM with 1 (500 ␮M; 0,
10, 20, 30, 60, 120, 180 min; 21 and 24 h) at a
detection wavelength of ␭ ϭ 280 nm with a
reference wavelength of 340 nm. Time 0 means 5
min preincubation without the NADPH-generat-
ing system. The chromatograms are representa-
tive for a series of three experiments.
After workup, supernatants of the incubations were analyzed by
Oxidative Metabolism of 1. Incubations of ARLM with 1 pro-
reversed-phase HPLC/DAD. Peaks of metabolites were separated and duced the widest spectrum of metabolites, with 2 through 8 displaying
examined using HPLC/MS and 1H NMR spectroscopy. Formation of the largest peak areas at a substrate concentration of 500 ␮M (Fig. 1).
the main metabolites and a decrease in substrate concentration were These metabolites were identified by comparison of HPLC retention
monitored in microsomal incubations of different species and differ- times and UV spectra to those of synthetic reference compounds, if
ent concentrations. The total content of P450 enzymes in these incu- available.
bations was determined to be 1.67 (ARLM), 0.33 (RLM), 0.88
(BLM), and 0.35 (HLM) nmol/mg protein.
The favored metabolic steps in ARLM were the hydroxylation of
the propenyl side chain leading to 2 and the hydroxylation at position
FIG. 2. Quantification of metabolite formation
during incubation of liver microsomes with 1:
A, ARLM (rat, Aroclor-induced); B, RLM (rat
noninduced); C, BLM (bovine); D, HLM (hu-
man). Initial concentration of 1 was 500 ␮M;
concentration of 10 (internal standard) was 500
␮M. Values are means Ϯ S.D. from three in-
dependent incubations. ϫ, sum of metabolites;
Ⅺ, 1; ‚, 2; छ, 5; ࡗ, 8; ꢀ, 3 ϩ 4 ϩ 6 ϩ 7.

METABOLISM OF METHYLISOEUGENOL IN LIVER
1731
TABLE 1
Amounts of substrate, internal standard, and metabolites detected in microsomal supernatants 2 h (or 10 min if indicated) after start of incubation
The amounts are expressed as percentage of the initial concentration of 1, which was 100 or 500 ␮M as indicated. Data represent means Ϯ S.D. from three incubations.
ARLM
100 ␮M (10 min)
RLM
BLM
HLM
Compound
500 ␮M
100 ␮M
500 ␮M
100 ␮M
500 ␮M
100 ␮M
500 ␮M
Internal standard (10)
Methylisoeugenol (1)
97.8 Ϯ 1.7
11.4 Ϯ 1.5
14.2 Ϯ 0.4
5.8 Ϯ 0.0
34.5 Ϯ 0.9
0.3 Ϯ 0.0
2.8 Ϯ 0.6
ND
101.8 Ϯ 5.9
24.7 Ϯ 5.0
12.6 Ϯ 1.2
6.9 Ϯ 3.5
15.6 Ϯ 0.9
0.5 Ϯ 0.2
3.2 Ϯ 1.1
12.1 Ϯ 5.5
50.9 Ϯ 3.2
99.6 Ϯ 2.0 101.5 Ϯ 5.4
99.3 Ϯ 3.4
8.3 Ϯ 0.4
24.2 Ϯ 1.5
1.8 Ϯ 0.2
3.3 Ϯ 1.1
2.7 Ϯ 0.7
2.2 Ϯ 0.1
ND
98.5 Ϯ 4.1
57.4 Ϯ 6.1
19.8 Ϯ 0.8
1.2 Ϯ 0.0
7.5 Ϯ 0.2
0.7 0.2
2.3 Ϯ 0.1
ND
31.5 Ϯ 17.1
100.8 Ϯ 3.0 100.6 Ϯ 2.0
36.5 Ϯ 5.3
26.2 Ϯ 7.4
4.5 Ϯ 0.2
ND
50.3 Ϯ 15.4
39.7 Ϯ 7.7
2.7 Ϯ 0.6
ND
ND
52.7 Ϯ 5.7
30.1 Ϯ 2.5
3.6 Ϯ 0.3
ND
3Ј-Hydroxymethyleugenol (2)
Isoeugenol ϩ isochavibetol (3 ϩ 4)
6-Hydroxymethylisoeugenol (5)
3Ј-Oxomethylisoeugenol (6)
1Ј,2Ј-Dihydroxymethylisoeugenol (7)
6-Hydroxymethyleugenol (8)
Sum of metabolites
33.9 Ϯ 3.2
5.0 Ϯ 0.4
ND
9.0 Ϯ 1.1
ND
2.1 Ϯ 0.0
ND
7.0 Ϯ 3.1
7.0 Ϯ 1.6
ND
3.0 Ϯ 1.1
1.0 Ϯ 0.1
ND
ND
ND
57.6 Ϯ 2.7
39.7 Ϯ 2.8
44.6 Ϯ 18.9
34.2 Ϯ 2.8
52.9 Ϯ 4.2
37.6 Ϯ 17.4
ND, not detectable.
6 of the aromatic ring leading to 5 and 8. Furthermore, the 1Ј,2Ј-diol several metabolites were below the limits of detection at earlier time
7 was identified. The products of demethylation of one of the methoxy points. In contrast, the metabolic capacity of ARLM was sufficient to
groups were identified as 3 and characterized as isochavibetol (4). lead to an almost complete loss of metabolites after 2 h. Therefore,
Although no reference compound was available for 4, its formation in
the incubation mixtures with liver microsomes was tentatively con-
firmed by analyzing its MS data ([MϩH]ϩ ϭ 165 m/z). Furthermore,
its UV spectrum [215 (1200 units), 260 (800 units), 300 (450 units)
nm] was identical (data not shown) to the spectrum of 3, as expected.
It is interesting to note that 5 could already be detected 10 min after
NADPH addition, whereas 8 was first detected 30 min after addition
of the NADPH-generating system. This metabolic transformation was
only found in incubations with ARLM. However, the concentration of
5 decreased during further incubation; that is, 5 could barely be
detected after 24 h of incubation. After 2 h, the formation of 6 became
observable as indicated by a ”negative” peak. This is due to the used
reference wavelength (340 nm) at which 6 exclusively shows a
marked absorption. Quantification of this metabolite was achieved
using a detection wavelength of 340 nm and a reference wavelength
of 450 nm.
In Fig. 2, the time course of levels of metabolites formed during
incubations of 2 (500 ␮M) with liver microsomes of different origins
is shown. The remaining substrate 1 and the major metabolites 2
through 7 could be quantified. For clarity of the figures, the concen-
trations of the metabolites 3, 4, 6, and 7 are shown as sums. The fact
that the calculated sum of concentrations of substrate and quantified
metabolites was less than 100% points to the formation of further
metabolites or reaction products, the amounts of which could not be
specified. This difference was found to increase with progressive time
of incubation, suggesting the formation of conjugated metabolites
and/or of metabolites binding to macromolecules. In ARLM (Fig.
2A), the strongest decline of 1 over time was observed. The 3Ј-alcohol
2, the ring-hydroxylated products 5 and 8, and the sum of the minor
metabolites 3, 4, and 7 almost equally contributed to the overall yield
of metabolites. In contrast, the slightly lower yield of metabolites in
microsomes from untreated rats (RLM) mainly consisted of 2 (Fig.
2B) with some minor formation of 3 ϩ 4 and 6 but a lack of 5, 7, and
8. In BLM 2 also dominated, whereas lower amounts of 5, 6, and 7
and of the sum of 3 ؉ 4 were found (Fig. 2C). In human liver
microsomes (Fig. 2D), a similar picture was obtained with respect to
the dominating role of 2. However, in addition to some formation of
3 ϩ 4 and 7, no formation of 5 or 8 could be detected.
In Table 1, the amounts of metabolites formed in ARLM, RLM,
BLM, and HLM are expressed as percentage of the substrate concen-
tration of 1 (at 100 or 500 ␮M starting concentration, respectively) at
FIG. 3. HPLC separation of ARLM incubation with 2 (3 h, 500 ␮M). Detection of
the start of the incubation. At the lower substrate concentration of 100
absorbance of eluting compounds at ␭ ϭ 261 nm (A), ␭ ϭ 280 nm (B). The
␮M, only levels after 2 h of incubation were monitored because chromatogram is representative for a series of three experiments.

1732
CARTUS ET AL.
FIG. 4. Formation of metabolites and decrease
of 5 during incubation of HLM with 5 (100
␮M; 10, 30, 60, 120 min) together with a
NADPH-generating system at
a detection
wavelength of ␭ ϭ 280 nm with a reference
wavelength of 340 nm. The chromatograms are
representative for a series of three experiments.
Table 1 shows the pattern of metabolites for ARLM at 100 ␮M oxo compound. According to their retention times and spectral properties,
substrate concentration after only 10 min. At a substrate concentration both of these compounds are presumed to be 13 (tR ϭ 10.5 min) and 14
of 100 ␮M, 5 became the dominating metabolite in ARLM, followed (tR ϭ 6.3 min). In contrast, the compounds with tR values of 11.7 and
by 2 and the minor metabolites 3, 4, 7, and 8. In RLM, the relative 12.3 min displayed strong absorbance at 261 nm and weaker absorbance
yield of 2 was also decreased at the lower substrate concentration; at 280 nm, being characteristic for compounds carrying a 2-propenyl side
however, this was not the case in BLM and HLM. In HLM, the chain. The retention times point to dihydroxy derivatives of 1 (e.g.,
decrease in substrate concentration even seemed to slightly enhance bearing one hydroxyl group at the side chain and another at the aromatic
the relative formation of 2, whereas formation of 7 was strongly ring). However, preparative separation of these secondary metabolites
enhanced.
failed, so identification via NMR spectra was not feasible.
Incubation of Synthesized Metabolites 2 and 5 with Liver Mi-
Because the formation of 8, representing an allylic compound,
crosomes. For the elucidation of the metabolic pathways, and for a during incubation of ARLM with 1 seems to be uncommon, we also
more precise specification of minor metabolites generated during investigated the metabolism of synthetic 5. In Fig. 4, chromatograms
incubations with 1, ARLM were incubated with compound 2, synthe- of incubation supernatants of HLM with 5 (100 ␮M, 2 h, tR ϭ 21.5
sized as a reference substance. After workup, the supernatants of these min) are depicted. The concentration of 8 (tR ϭ 20.0 min) continu-
incubations were applied to HPLC analysis.
ously increased during the incubation period. At a retention time of
Five secondary metabolites were detected in addition to nonme- 16.3 min, a metabolite was formed displaying a negative peak. The
tabolized substrate (57%) after 3 h (Fig. 3). The negative peak eluting UV spectrum of this compound showed a maximum of absorption at
at 19 min was identified as the aldehyde 6, resulting from microsomal 340 nm, indicative of an ␣,␤-unsaturated oxo compound. The meta-
oxidation of the hydroxy group in the 3Ј position. The compound with a bolic transformation of 5 leading to 8 was observed for all liver
tR of 10.5 min showed intense absorption at 280 nm (Fig. 3A) and a microsomes tested using 100 ␮M starting concentration of 5 with a
weaker absorption at 261 nm (Fig. 3B), more characteristic for an allyl or ranking HLM Ͼ ARLM Ͼ BLM Ͼ RLM (data not shown). However,
saturated side chain attached to the phenyl ring. The peak at a tR of 6.3 using higher starting concentrations of 5 (500 ␮M and 1 mM), the
min displayed spectroscopic characteristics indicating an ␣,␤-unsaturated formation rate of 5 rapidly decreases (data not shown).
FIG. 5. Phase I metabolic pathways of 1 in
liver microsomes.

METABOLISM OF METHYLISOEUGENOL IN LIVER
1733
Discussion
investigation is needed to determine whether the differences found in
liver microsomes from three different species are indicative of species-
selective patterns of hepatic metabolism of 1 in intact cells or in vivo.
3Ј-Hydroxylation forming 2 was the major pathway of 1. Alcohol
2 was shown to be the precursor of the ␣,␤-unsaturated aldehyde 6.
This metabolite and the 1Ј,2Ј-epoxide 12, which could not be detected
but can be assumed to be a precursor of the 1Ј,2Ј-diol 7, have the
reactive capacity to bind to proteins and other hepatic macromole-
cules. The second major reaction of 1 in bovine and Aroclor1254-
induced rat liver microsomes was found to be ring hydroxylation at
position 6. This reaction was not or barely detectable in microsomes
from humans and noninduced rats. We were surprised to find that
formation of 8, the allylic isomer of the 6-hydroxylated metabolite 5,
was observed, bringing about the capability of formation of putative
genotoxic secondary metabolites hydroxylated at the 1Ј position.
Future studies will be aimed at investigating the actual toxic potency
of these reactive metabolites and their relevance of formation.
Whereas previous investigations on incubations of liver micro-
somes treated with 11 as a substrate (Gardner et al., 1997; Smith et al.,
2002; Jeurissen et al., 2006) were performed to clarify the formation
of the proximate carcinogen 9, no reports dealing with investigations
on the metabolism of 1 in liver microsomes are available.
Incubation with microsomes from untreated and Aroclor1254-
treated rats and of bovine and human origin revealed the more or less
complex pattern of phase-I metabolites depicted in Fig. 5. As dis-
cussed in the following, some of the metabolites shown were not
found in all incubations.
The main metabolite in nearly all types of incubations, the 3Ј-
hydroxy metabolite 2 is considered as a nonmutagenic product. How-
ever, our study revealed that it can be converted into the ␣,␤-unsat-
urated aldehyde 6 and is probably capable of forming Michael
adducts. Such reactions with, for example, amino acids and proteins,
possibly play a role in liver toxicity and hapten formation. 6 is likely
to be metabolized further to the corresponding carboxylic acid 14.
In contrast to incubations with methyleugenol, whereupon isomer-
ization of the side chain and hydroxylation in the 3Ј position was
found (A. T. Cartus, unpublished data), the corresponding isomeriza-
tion of 2 resulting in the formation of 9 was not observed in our
experiments. This is in agreement with previous investigations that
showed a lack of genotoxicity of 1 in rat hepatocytes in culture
(Hasheminejad and Caldwell, 1994). Starting with a propen-1-yl side
chain, the formation of an allylic cation obviously does not seem to be
energetically advantageous. However, as early as 30 min after starting
the ARLM incubation with 1, the formation of 8 was observed,
suggesting that the hydroxy group adjacent to the propenyl side chain
may facilitate isomerization to the corresponding allyl compound.
Similar findings have been reported for the metabolism of asarones.
Acknowledgments
We thank Dr. Hans-Joachim Schmitz for providing rat livers, Eva
Gorgus for technical support, and Christiane Mu¨ller for the measurement of
NMR spectra. The provision of bovine liver microsomes by the team of
Professor Gerhard Eisenbrand at the University of Kaiserslautern is grate-
fully acknowledged.
Authorship Contributions
Participated in research design: Cartus and Schrenk.
Conducted experiments: Cartus.
Contributed new reagents or analytic tools: Cartus and Merz.
Performed data analysis: Cartus.
Wrote or contributed to the writing of the manuscript: Cartus, Merz, and
Schrenk.
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noninduced RLM.
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These findings show that microsomes from human, rat, and bovine
liver are able to generate a broad spectrum of phase-I metabolites
when incubated with 1, a common ingredient of cosmetics and con-
stituent of flavoring preparations in foods and beverages. However, an
Address correspondence to: Prof. Dr. Dieter Schrenk, Food Chemistry and
Toxicology, University of Kaiserslautern, Erwin-Schroedinger-Strasse, D-67663
Kaiserslautern, Germany. E-mail: schrenk@rhrk.uni-kl.de