Analysis of the content of the diterpenes cafestol

Food and Chemical Toxicology 35 (1997) 547-554
Analysis of the Content of the Diterpenes
Cafestol and Kahweol in Coffee Brews
G . G R O S S , E. J A C C A U D a n d A. C. H U G G E T T *
Department of Quality and Safety Assurance, Nestl6 Research Centre, Vers-chez-les-Blanc, PC Box 44,
CH-1000 Lausanne 26, Switzerland
(Accepted 30 July 1996)
Abstract--The diterpenes cafestol and kahweol have been implicated as the components in boiled coffee
responsible for its hypercholesterolaemic effects. These particular coffee constituents have also been
shown to possess anticarcinogenic effects. A simple and sensitive reverse-phase HPLC method using
solid-phase extraction has been developed for the analysis of cafestol and kahweol in coffee brews. This
method was used to confirm that the method of coffee brewing is a major determinant of the cup content and hence level of consumption of these diterpenes. Scandinavian-style boiled coffee and Turkishstyle coffee contained the highest amounts, equivalent to 7.2 and 5.3 mg cafestol per cup and 7.2 and
5.4 mg kahweol per cup, respectively. In contrast, instant and drip-filtered coffee brews contained negligible amounts of these diterpenes, and espresso coffee contained intermediate amounts, about 1 mg
cafestol and 1 mg kahweol per cup. These findings provide an explanation for the hypercholesterolaemic effect previously observed for boiled coffee and Turkish-style coffee, and the lack of effect of instant
or drip-filtered coffee brews. This methodology will be of value in more correctly assessing the human
exposure to these diterpenes through the consumption of coffee, and hence the potential physiological
effects of different brews. © 1997 Elsevier Science Ltd
AbbreviatioJts: C = cafestol; K = kahweol; C + K = cafestol and kahweol; C + K/P = cafestol and
kahweol palmitates.
Ever since the introduction of coffee into Europe in
the 17th century ~:here has been interest concerning
its possible beneficial or negative effects on human
health. Recently, attention has focused on the biological effects of the major diterpenes present in coffee--cafestol and kahweol (Di Simplicio et al.,
1989; Lam et al., 1982; Mensink et al., 1995; Miller
et al., 1991; Wattenberg et al., 1985; Weusten-Van
der Wouw et al., 1994). These components, which
appear to be rela~fively specific to coffee, are found
in both Arabica and Robusta (predominantly cafestol) varieties (Lercker et al., 1995) and comprise up
to about 10-15% of the lipidic fraction of roasted
coffee beans (Ratnayake et al., 1993). Although the
total diterpene content of various brewed coffees
has been reported previously (Ratnayake et al.,
1993), there has been only one report on the specific
content of cafestol and kahweol in different brews
(Urgert et al., 1995b).
The consumption of 'boiled' coffee, a brew
peculiar to Scandinavian countries, has been shown
to be associated with elevated levels of serum cholesterol (Pietinen et al., 1990; Stensvold et al., 1989;
*Author for correspondence.
Thelle et al., 1983 and 1987). Epidemiological and
controlled clinical studies have indicated that this
hypercholesterolaemic effect is dependent on the
method of preparation of the coffee brew (Arc
et al., 1987; Bak and Grobbee, 1989; Theile and
Van der Stegen, 1990). For example, in contrast to
'boiled' coffee, the consumption of instant or filtered coffee has no significant effect on serum cholesterol levels (Burr et al., 1989, Van Dusseldorp
et al., 1991), whereas Turkish or Middle Easternstyle coffees appear to increase serum cholesterol
(Kark et al., 1985). A recent series of clinical trials
have confirmed that the hypercholesterolaemic
agents are present in the lipid fraction of 'boiled'
coffee and have identified the major causative
agents as the diterpenes cafestol and kahweol which
are present mainly as fatty acid esters (WeustenVan der Wouw et al., 1994). These investigations
demonstrated a dose-response effect of cafestol and
kahweol on increasing serum cholesterol levels.
Feeding studies in rodents using coffee oils and purified coffee diterpenes have confirmed the hypercholesterolaemic effects of these compounds (Huggett
et al., 1993; Ratnayake et al., 1995; Sanders and
Sandaradura, 1992). However, the sensitivity of experimental animals to the hypercholesterolaemic
effects of coffee diterpenes appears to be modulated
0278-6915/97/$17.00 + 0.00 © 1997 Elsevier Science Ltd. All rights reserved. Printed in Great Britain
PII S0278-69 i 5(96)00123-8
G. Gross et al.
by the diet composition (Ratnayake et al., 1995),
which may explain why some investigators were
unable to demonstrate a cholesterol-raising effect of
boiled coffee (Mensink et al., 1992).
Coffee has been shown to contain cancer chemopreventive agents (Wattenberg, 1983). Aeschbacher
and Jaccaud (1990) demonstrated that oral administration of coffee to mice decreased nitrosoureamediated D N A damage. When added to the diet of
experimental animals, coffee inhibited the development of 7,12-dimethylbenz[a]anthracene (DMBA)induced oral carcinomas and mammary tumours
(Miller et al., 1988 and 1993; Wattenberg, 1983).
Further studies demonstrated that a (50:50) mixture
of cafestol and kahweol produced a similar inhibition of buccal pouch tumours (Miller et al.,
1991). The mechanisms responsible for the anticarcinogenic effects of cafestol and kahweol have not
been elucidated; however, these compounds have
been shown to be potent inducers of glutathione Stransferases (Di Simplicio et al., 1989; Huggett and
Schilter, 1995; Lam et al., 1982). The enhancement
of these activities may play a role in the chemopreventive action of the diterpenes by catalysing the
detoxification of reactive carcinogenic electrophiles.
Information on the content of cafestol and kahweol in coffee brews allows a prediction of the
likely effects of different coffee brews on serum cholesterol levels and hence their potential role in cardiovascular disease, and also provides a basis for
determining the relevance of these biologically
active constituents with regard to the reported chemoprotective effects of coffee (Baron et al., 1994).
Although methods for the analysis of total coffee
diterpenes have been reported, these are of inadequate sensitivity or specificity for application to
the measurement of individual diterpenes in coffee
brews (Ratnayake et al., 1993). We report the development of a simple and sensitive reverse-phase
HPLC method using solid-phase extraction procedures that has been applied to the analysis of
cafestol and kahweol in various coffee brews. This
methodology complements the gas chromatographic
method for these components reported recently by
Urgert et al. (1995b) and permits a corroboration
of the finding that the method of preparation of the
brew is a critical determining factor in determining
the daily intake of these diterpenes from coffee consumption.
Materials and reagents
All solvents used were of analytical or HPLC
grade. If not otherwise stated, the water used was
18 MfLcm quality obtained from a Milli-Q water
purification system (Millipore, Bedford, MA, USA).
A mixture of 17-O-palmitoyl-cafestol (52.5%, w/w)
a n d 17-O-palmitoyl-kahweol (47.5%, w/w), hereafter referred to as C + K/P, prepared according to
the method of Bertholet (1987), was used as refer-
ence standard. Its purity was checked by saponification and quantification of liberated diterpene
alcohols, cafestol (C) and kahweol (K), by HPLC
using the method described below. Major by-products in C + K/P were identified as isokahweolene
(0.3%, w/w) and cafestolene (0.2%, w/w). Free
cafestol and kahweol reference standard mixture
[54.5% (w/w) cafestol; 45.5% (w/w) kahweol], hereafter referred to as C + K, was prepared from coffee oil as previously reported (Bertholet, 1987).
C + K purity was confirmed by mass spectroscopy
as well as by proton and carbon-13 nuclear magnetic resonance spectroscopy. Stock solutions of
C + K/P in hexane (2.5mg/ml), and C + K in
ethanol (0.5, 2.5 and 5 mg/ml) were prepared.
Extrelut extraction cartridges (3 ml) were from
Merck Ltd (Darmstadt, Germany). Bond-Elut PRS
(propyl sulfonic acid; 100rag) and C18 (100rag)
cartridges and cartridge coupling adapters were
from Analytichem International (ICT AG, Basle,
Switzerland). Purified extracts were stored in 1.1 ml
Chromacol gold microvials (Infochroma AG, Zug,
Solid-phase extraction cartridge conditioning
C18 cartridges were rinsed with 3 ml 2-propanolethyl acetate (1:1). PRS cartridges were rinsed with,
in order, 3 ml 1 y hydrochloric acid solution, 20 ml
water and 3 ml 2-propanol-ethyl acetate (1:1). One
C18 was coupled to a PRS cartridge using an
adapter piece. This assembly is hereafter referred to
as 'tandem'.
Chromatographic analysis was performed using a
Merck L-6200 pump, a Gilson 231-401 automated
sample processor and injector, and a Hewlett
Packard model 1040A/mark II multi-channel ultraviolet detector driven by a HP9000 series 300 workstation (Chemstation). A 250 x 4.6 mm Merck
Superspher Lichrocart 100 RP-18 column (3/~m
particle size) was used to separate the diterpenes.
The mobile phase consisted of water (solvent A)
and methanol (solvent B). Separations were carried
out at ambient temperature using linear gradient
elution conditions (0 rain: 30% A, 70% B; 20 rain.
5% A, 95% B) and a flow rate of 1 ml/min. C and
K were detected at 230 nm and 290 nm, respectively. Peaks were quantified by area measurement
and identified by retention times and by matching
on-line recorded spectra with library ultraviolet
spectra. Injections were performed in the partial
loop filling mode using a custom program for the
Gilson '231-401 injector. In general, a 20 ml portion
of purified extract or a 5 pl standard preparation
w a s injected into a 50 pl loop, followed by dilutant
liquid (20/A) to compensate for the injector port
dead volume.
The HPLC instrument was calibrated on a
weekly basis by injecting each C + K stock solution.
Cafestol and kahweol in coffee brews
Preparation of coffee brews
All coffee brews were prepared using a commercial Arabica blend of roasted coffee purchased in
Switzerland, except for single-serving size espresso
brews which were purchased as prepacked capsules
containing different blends, and instant coffee,
which was prepared from two different commercial
soluble coffee blends. For preparation of 'boiled
coffee', coffee beans were coarsely ground (800900 pm), whereas for other coffee brews the coffee
beans were ground to the same medium-fine particle
size (500-550 pm). Tap water was used to prepare
all coffee brews. Brewing methods, brew strengths
and cup sizes were standardized according to IARC
Boiled coffee. Was prepared in the traditional
manner by boiling 9 g coarsely ground roasted coffee with 150 ml boiling water for 10 min. The mixture was hot-filtered through a metal screen (mesh
size 0.5 mm) and the filtrate used for analysis.
Turkish coffee. Was prepared by bringing to a
gentle boil a mixture of 5 g roast and ground coffee,
10 g saccharose and 60 ml cold water. Boiling was
interrupted when a foam developed on the surface
of the brew. The brew was left to settle and the
supernatant was taken for analysis.
Mocha (moka, Neapolitan) coffee. Was prepared
using a commercial Italian coffee maker. The water
reservoir, filter cup and coffee recipient were separated and the filter cup filled with 20 g roasted coffee
powder. Cold water (300 ml) was added to the bottom reservoir, and the filter cup was inserted. The
coffee maker was reassembled and then heated on a
hotplate until the bottom water reservoir was
empty. The resulting coffee brew was mixed prior
to analysis.
Standard espresso coffee. Was prepared using a
mechanical-type Turmix EX10 coffee machine (Turmix Ltd, Jona, Switzerland). 20 g of roast and
ground coffee was used to draw a 130 ml volume.
Single-serving size espresso. Was prepared using a
Turmix CII01 coffee machine (Turmix Ltd, Jona,
Switzerland). One commercially available disposable
capsule containing 5 g roasted coffee was used to
draw a cup of about 50 ml. Five different commercial prepacked coffee blends were examined.
Drip-filtered coffee. Was prepared by adding 13 g
roast and ground coffee into a Tchibo No. 4 type
paper filter (Tchibo GmbH, Hamburg, Germany)
placed on an aplgropriate funnel. Boiling water
(200 ml) was added and the filtered brewed coffee
was collected.
Instant coffee. Was prepared by mixing 170 ml
boiling water with 2 g instant coffee granules per
cup. Two different commercial coffee blends were
Extraction procedure. The brewed coffees were
allowed to cool to room temperature and adjusted
to pH 9-10 using 4 N sodium hydroxide solution.
Aliquots of 2.5 ml were transferred into plastic test
tubes (Falcon 2059, Becton Dickinson, N J, USA)
and spiked with suitable quantities of C + K/P
standard solution using a graduated microlitre syringe (Hamilton 1705/1710, Hamilton Ltd, Bonaduz,
Switzerland). The resulting mixtures were vortex
mixed and applied to an Extrelut-3 columns placed
on a Supelco Visiprep ® vacuum manifold. The application of a slight vacuum accelerated the adsorption of the liquid phase onto the column material.
The Extrelut columns were then placed on a rack
and fine luer lock needles (25G x 5/8 inch) were
applied to the tips of the columns to serve as flow
restrictors. Each sample tube was rinsed with 2.5 ml
hexane-dichloromethane (95:5, v/v) which was then
poured into the respective Extrelut column. After
standing for approximately 15-30 minutes, the
Extrelut columns were eluted under gravity flow by
the addition of 12.5ml hexane-dichloromethane
(95:5) to the column reservoir. Approximately 11 ml
eluate per column was collected into 25 ml pearshaped flasks. The eluates were evaporated to dryness at ambient temperature under reduced pressure.
The resulting residues were saponified by the addition of 2 ml methanolic 0.3 N potassium hydroxide solution. Following incubation with agitation
for 60 min at room temperature using a model KL2
agitator (Universal Kleinstschtittler, Edmund
BiJhler GmbH, Tiibingen, Germany), 800/~1 2-propanol-ethyl acetate (1:1, v/v) and 800/~1 0.5 M aqueous sodium dihydrogen phosphate were added.
After thorough mixing, this preparation was transferred into the top reservoir of previously conditioned C18/PRS solid-phase extraction cartridge
tandems and slowly eluted through both cartridges
by applying gentle air pressure with a plastic syringe attached to the top of the tandem. The eluate
was collected in 16 x 76 mm poly-allomer test-tubes
(Beckman Instruments, Palo Alto, CA, USA). The
flask containing the incubation mixtures was rinsed
with 1 ml 2-propanol-ethyl acetate (1:1, v/v), and
the washings were passed through the tandem and
combined with the previous eluate. The combined
eluate was concentrated using a model SVCI00H
Speed-Vac vacuum centrifuge with a RT100A cryotrap (Savant Instruments, Farmingdale, NY, USA).
The residue was reconstituted in 400/~1 methanol
with vortex-mixing and then filtered through a
0.2pro zero dead volume Nalgene filter tip
(Nalgene Company, Rochester, NY, USA) into an
HPLC microvial. Samples were stored at +4°C
prior to HPLC analysis.
Quantitative analysis was performed using external standard calibrations. The method of standard
additions was employed to check C and K extraction efficiencies and the results were corrected
accordingly. In general, one preliminary determination was carried out to approximate the C and K
levels in the examined product. This was then followed by triplicate determinations using one
unspiked sample and two parallel samples spiked
G. Gross et al.
with the C + K/P mixture prior to the extraction
procedure so as to double and triple the estimated
levels of C and K. The extraction efficiencies were
calculated for both analyses as the slope of the linear regression line using the parameters added (x) v.
measured (y) analyte concentration. If not otherwise
stated, the results are expressed as concentrations of
free diterpene alcohols.
Optimization of analytical methodology
Diterpene alcohols were analysed by HPLC following extraction of the corresponding esters from
the coffee brews, saponification, and clean-up of the
saponified mixture using solid-phase extraction
technology. The method used is schematized in
Fig. 1. Initially, water/hexane partitioning of the
coffee brew was evaluated as a means to pre-concentrate the diterpene esters. However, this procedure was abandoned owing to technical
difficulties (i.e. formation of stable emulsions and
multiple liquid phases). The use of diatomaceous
earth (Extrelut ®) to immobilize the aqueous liquid
phase permitted the efficient extraction of diterpene
esters with hexane while avoiding the formation of
emulsions. The saponification of C and K proceeded smoothly at room temperature and was
complete within 1 hr. Longer hydrolysis times
diminished the yields, while much shorter times
resulted in incomplete saponification (data not
shown). The crude hydrolysate was purified using
Coffee brew pH 9
Partition on Extrelut-3
Extraction with hexane- DCM, 95:5
(IncreasedC + K palmitateextractibility)
Residual linidsremoval
Extraction withAcOEt: 2-propanol, 1:1
(IncreasedC + K extractibility)
Salts removal
HPLC analysis
Fig. 1. Outline of the procedure employed for the solidphase extraction of diterpenes from coffee brews:
DCM = dichloromethane; SPE = solid-phase extraction;
AcOEt = ethyl acetate; MeOH = methanol.
C18 silica to remove residual lipids, followed by a
propyl sulfonic acid cation exchanger to reduce the
salt content of the extracts. These two processes
were conveniently combined in a one-step procedure
by the use of coupled cartridges. Following reconstitution, the resulting extract could then be directly
injected into the liquid chromatograph.
The accuracy of the quantification of C and K
levels depends critically on the extraction efficiency
of the corresponding esters from crude coffee, the
yield of the saponification, and the recovery of free
C and K during the final filtration. In order to optimize these parameters, we used the method of standard additions. Employing the protocol described,
including the use of triplicate extractions, a set of
three data pairs [spiked concentration (x)/determined concentration (y)] was created for each analyte and the extraction efficiency expressed as the
slope of the respective linear regression line
(r 2 = 0.999 and r 2 = 0.997 for C and K, respectively). The mean extraction efficiencies were
97 + 19% (n = 16) for C and 96_+ 19% (n = 16)
for K.
The specificity of C and K determinations was
provided by the selective detection of both compounds at their respective wavelength of maximal
absorption, as well as the match of on-peak
recorded ultraviolet spectra with references. The
limit of quantification was estimated as approximately 0.05 mg/litre for both C and K, sufficiently
sensitive for the analysis of all of the coffee brews
investigated in the present study. The chromatograms obtained from analyses of an espresso blend
soluble coffee are shown in Fig. 2.
In our laboratory the method showed good
repeatability as demonstrated by the coefficient of
variation of 8.6% (C) and 8.7% (K) obtained following triplicate determinations of the same sample
(containing 17.3 mg C/litre and 16.3 mg K/litre, respectively).
Quantification of cafestol and kahweol m various coffee brews
C and K were analysed in seven different coffee
brews prepared using a variety of methods. The
results are summarized in Table 1.
Interestingly, Turkish and not boiled coffee contained the highest concentration of C and K.
However, when the results were expressed on the
basis of cup size, it was apparent that boiled coffee
(14.4mg/cup) provides more of these diterpenes
than Turkish-style coffee (10.7 mg/cup). The
amount of C and K measured in mocha
(Neopolitan)-style coffee was also relatively high,
about 75% of the concentrations found in boiled
coffee and about 32% when expressed as a ratio of
cup size (4.6 rag/cup). The C and K concentrations
found in standard espresso brewed coffee were
almost three times less than observed for boiled coffee, despite a brewing strength almost 2.5-fold
greater than that used for boiled coffee. When
expressed as the amount per cup, the C + K level
Cafestol and kahweol in coffee brews
230 nm
Reference spectrum
I~" Kahweol
250 30O 35O
290 nm
Time (min)
Fig. 2. Chromatographic profiles of espresso blend instant coffee recorded at 230 nm (top) and 290 nm
(bottom): A = unspiked sample containing 44 pg C/g and 40 #g K/g (values not corrected for incomplete extraction efficiency); B and C = profiles of the same sample spiked with 209/~gC/g and
171 pg K/g, and 418 #g C/g and 342 ttg K/g, respectively, prior to analysis; mAU = milli Absorbance
of espresso (2 mg/cup) was over seven times lower
than for boiled coffee. The C + K concentration of
espresso coffee brews prepared from single-serving
size prepacked roast and ground coffees was also
considerably lower than for boiled coffee.
Furthermore, the amount of C and K in these
espresso brews prepared from capsules (Table 1,
Blends A-E, 0.15-0.35mg C + K / c u p ) ranged
from about five to 14 times lower than for the
espressos prepared using the traditional method.
The blend of coffee employed in the capsules
appeared to influence the diterpene content of the
brew to some exterLt. Instant coffees contained very
low concentrations of C and K (0.2-0.5 rag/cup),
providing between about 25 and 70 times less
C + K per cup than boiled coffee. Drip-filtered cof-
fee contained the least C + K, equivalent to a total
of 0.04 mg per cup.
A simple and sensitive reverse-phase HPLC
method using solid-phase extraction has been developed for the analysis of C and K in coffee brews.
This method benefits from a straightforward extraction procedure that overcomes the problems of
emulsion formation and the presence of multiple
liquid phases encountered when using direct solvent
extractions of coffee. The method of standard additions was selected in place of an internal standard
for quantification, since an appropriate internal
standard was not available. An internal standard
G. Gross et al.
Table 1. Analysis of diterpene alcohols in coffee brews
Diterpene alcohols
Brew strength (mg/ml)
Coffee brew
mg/litre+ SD
mg/litre + SD
Turkish coffee
Boiled coffee
Mocha coffee
88.7 ± 4.0
48.3 + 3.8
37.5 + 1.3
89.9 + 4.1
48 + 2.5
38.5 + 0.9
Espresso coffee
Exp. 1
Exp. 2
17.3 ± 1.5
16.3 + 1.4
17.1 +0.5
Single-servingsize espresso
Blend A
Blend B
Blend C
Blend D
Blend E
Filtered coffee 25-75
3.4 + 0.3
2.1 _+0.3
2.2 + 0.2
1.6 + 0.7
1.2 + 0.3
0.12 + 0.02
3.5 + 0.3
2.9 + 0.2
2.6 ± 0.3
1.8 + 0.8
1.7 + 0.3
0.14 + 0.03
Instant coffee
Standard blend
Espresso blend
0.7 + 0.07
0.7 + 0.10
10 16
*Coffee cup sizes are as follows: 150 ml for boiled, filtered and instant coffees; 60 ml for Turkish, mocha and espresso
coffees; and 50 ml for the single serving size espresso coffees.
selected purely on the basis of its chromatographic
behaviour (Urgert et al., 1995b) suffers from an inability to take into account the variability that may
occur in saponification or extraction efficiencies.
Turkish-style coffee, and not boiled coffee, contained the highest concentration of C and K. A
similar result was previously reported by Ratnayake
et al. (1993) and this may be a consequence of the
higher amount of fines present in Turkish-style
coffee, compared with 'boiled coffee'. Thus the
method by which 'boiled coffee' is decanted may
have a large influence on its diterpene content.
Furthermore, increasing the time of boiling did not
increase the C and K contents of the 'boiled coffee'
(data not shown). Similar findings have been
reported by Urgert et al. (1995b), who observed
extremely large variations in field samples of these
coffee brews and attributed this variation to differences in brewing strength and the amount of bean
particles decanted with the brew. They also
reported that field samples of Scandinavian boiled
and Turkish/Greek coffees contained between 1 and
10mg cafestol per cup (equivalent to about 220 mg/cup) of C + K. The values that we obtained
for a standardized boiled coffee (14.4 mg/cup) and
Turkish coffee (10.7 mg/cup) prepared under laboratory conditions from the same roast and ground
coffee beans according to IARC (1991) fall within
this range. The C + K content for mocha
(Neopolitan)-style coffee (4.6mg/cup) is a little
higher than that previously reported by Urgert et al.
(1995b) for a laboratory-prepared brew (2.5mg/
cup); nevertheless, in both studies this brewing
method results in a coffee providing intermediate
amounts of C and K. It is interesting to note that it
was recently reported that consumption of three
cups per day of Italian brewed coffee (moka,
mocha) for 5 wk in a double-blind crossover clinical
trial had no significant effect on serum cholesterol
levels (Sanguigni et al., 1995).
The C and K concentrations found in standard
espresso brewed coffee (2 mg/cup) is consistent with
the field samples (about 0-6 mg/cup) reported elsewhere (Urgert et al., 1995b). However, this finding
is in contrast to that reported by Ratnayake et al.
(1993) which indicated that espresso coffee contained twice the level of total diterpenes compared
to 'boiled' and Turkish coffees. The reason for this
discrepancy is unclear, but it is likely that diterpene
levels may be influenced by steam pressure, contact
time of steam with grounds, and the efficacy of the
metal screen strainer in retaining fine coffee particles, thereby preventing their contribution to the
diterpene content of the brewed coffee. The C + K
contents of coffee brews prepared from single-serving size prepacked roast and ground coffees (0.150.35 mg/cup) were considerably lower than those of
traditional espresso coffee. The reason for this
difference is not obviously apparent but it seems
likely that the prepacked capsules act as a filter,
thus allowing fewer fine particles of coffee grounds
to pass into the brew. This explanation is consistent
with a recent report indicating that spent fine coffee
grounds raise serum cholesterol levels when administered to human volunteers, indicating that the
fines are an important source of diterpenes (Urgert
et al., 1995a).
Both drip-filtered and instant coffees contained
very low concentrations of C + K, a finding which
is in good agreement with that reported by Urgert
et al. (1995b) and also with the low levels of total
lipids and total diterpenes found by Ratnayake et al.
(1993) for these types of coffee. The low levels of
diterpene esters in drip-filtered coffee prepared
Cafestol and kahweol in coffee brews
1.5 --
= 1.0
0.5 -
s' r' so,I
C + K (mg/day)
Fig. 3. Predicted increase in serum total cholesterol levels
following the consunaption of five cups/day of different
coffee brews for 4 wk. The dose-response curve is based
upon data points (O) taken from studies in human volunteers previously repo:rted by Weusten-Van der Wouw et al.
(1994). The estimated C + K intakes are based upon the
consumption of five cups of coffee brews of the following
sizes: boiled, filtered, instant--150ml; Turkish, mocha,
espresso--60 ml; prepacked espresso capsules--50 ml. The
predicted increase in serum cholesterol is less than
0.1 mmol/litre for coffee brews (single-serving size
espresso, instant, filtered) not shown in the figure.
using a paper filter can be explained by both the
removal of fines and the strong lipid-binding effect
of filter paper (Ahola et al., 1991). For instant coffee, a large proportion of the diterpenes is lost
during industrial processing.
The potential cholesterol-raising effects of the
different coffee brews can be estimated on the basis
of the results of clinical studies in which 'boiled coffee', coffee oils or purified diterpenes were administered to volunteers, as reported by Weusten-Van
der W o u w et al. (1994). A review of these studies
demonstrates a dose-dependent effect of the daily
ingestion of preparations containing C and K for
4 wk on the increase in serum cholesterol levels
(Fig. 3). If it is assumed that five cups of coffee are
consumed daily, oll the basis of the results reported
in the present investigation, the expected increase in
cholesterol levels would range from less than
for filtered coffee to about
0.85 mmol/litre for boiled coffee. It must be emphasized that this extrapolation is based on assumptions and uncertainities concerning the diterpene
contents of some of the test materials used in the
clinical studies. Nevertheless, it is interesting to note
that Pietinen et al. (1990) reported that Finnish
men and women who regularly drank seven to nine
cups of boiled coffee per day had serum cholesterol
levels 0.6 mmol/litre higher than those of consumers
of filtered coffee. Furthermore, a cross-sectional
study in Norwegians found that subjects habitually
consuming five or more cups of boiled coffee per
day had mean serum cholesterol levels 0.3 mmol/
litre higher than those of matched filter coffee drinkers. (Weusten-Van der W o u w et al., 1994).
In conclusion, the use of solid-phase extraction
techniques eliminates the problems of multiple
phase separation that are often encountered when
liquid-liquid extraction techniques are used with
coffee brews. The results for C and K in different
coffee brews are consistent with those previously
reported and with the results predicted from an
evaluation of clinical and epidemiological studies
comparing the hypercholesterolaemic effects of
different brews. Finally, our fndings indicate that
the levels of C and K found in traditional espresso
coffees are lower than previously suspected and predict that instant and filtered coffees would not have
any effect on serum cholesterol levels. This new
methodology should help to assess more correctly
the exposure to these compounds through the consumption of coffee and to predict more accurately
the physiological effects of different coffee brews.
Aeschbacher H. U. and Jaccaud E. (1990) Inhibition by
coffee of nitrosourea mediated DNA damage in mice.
Food and Chemical Toxicology 28, 633-637.
Ahola I., Jauhiainen M. and Aro A. (1991) The hypercholesterolaemic factor of boiled coffee is retained by a
paper filter. Journal of Internal Medicine 230, 293-297.
Aro A., Tuomilehto J., Kostiainen E., Uusitalo U. and
Pietinen P. (1987) Boiled coffee increases serum low
density lipoprotein concentration. Metabolism 36, 10271030.
Bak A. A. A. and Grobbee D. E. (1989) The effect on
serum cholesterol levels of coffee brewed by filtering or
boiling. New England Journal of Medicine 321, 14321437.
Baron J. A., de Verdier M. G. and Ekbom A. (1994)
Coffee, tea, tobacco, and cancer of the large bowel.
Cancer Epidemiology, Biomarkers and Prevention 3, 565570.
Bertholet R. (1987) Preparation of cafestol. United States
Patent 4,692,534.
Burr M. L., Gallacher J. E., Butland B. K., Bolton
C. H. and Downs L. G. (1989) Coffee, blood pressure
and plasma lipids: a randomized controlled trial.
European Journal of Clinical Nutrition 43, 477-483.
Di Simplicio P., Jensson H. and Mannervik B. (1989)
Effects of inducers of drug metabolism on basic hepatic
forms of mouse glutathione transferase. Biochemical
Journal 263, 679-685.
Huggett A. C., Marchesini M. and Perrin I. (1993) Effects
of coffee oil on plasma cholesterol in the rat. In 15th
International Scient!fic Colloquium on Coffee. pp. 497503. Association Scientifique International du Caf&
Huggett A. C. and Schilter B. (1995) The chemoprotective
effects of cafestol and kahweol: effects on xenobiotic
metabolising enzymes. Journal of Cellular Biochemistry
19A, 190.
IARC (1991) IARC Monographs on the Evaluation of the
Carcinogenic Risks to Humans. Vol. 51. Coffee, Tea,
Mat~, Methylxanthines and Methylglyoxal. pp. 41-206.
International Agency for Research on Cancer, Lyon.
Kark J. D., Friedlander Y., Kaufmann N. A. and Stein
Y. (1985) Coffee, tea and plasma cholesterol: the
Jerusalem Lipid Research Clinic prevalence study.
British Medical Journal 291, 699-704.
Lam L. T. K., Sparnins V. L. and Wattenberg L. W. (1982)
Isolation and identification of kahweol palmitate and
cafestol palmitate as active constituents of green coffee
beans that enhance glutathione S-transferase activity in
the mouse. Cancer Research 42, 1193-1198.
Lercker G., Frega N., Bocci F. and Rodriguez-Estrada
M. T. (1995) High resolution gas chromatographic determination of diterpenic alcohols and sterols in coffee
lipids. Chromatographia 41, 29-33.
G. Gross et al.
Mensink R. P., Lebbink W. J., Lobbezoo 1. E., WeustenVan der Wouw M. P. M. E., Zock P. L. and Katan
M. B. (1995) Diterpene composition of oils from
Arabica and Robusta coffee beans and their effects on
serum lipids in man. Journal of Internal Medicine 237,
Mensink R. P., Zock P. L., Katan M. B. and Beynen
A. C. (1992) Boiled coffee does not increase serum cholesterol in gerbils and hamsters. Zeitschrift far
Ernahrungswissenschaft 31, 82-85.
Miller E. G., Formby W. A., Rivera-Hidalgo F. and
Wright J. M. (1988) Inhibition of hamster buccal pouch
carcinogenesis by green coffee beans. Oral Surgery 65,
Miller E. G., Gonzales-Sanders A. P., Couvillon A. M.,
Binnie W. H., Sunahara G. I. and Bertholet R. (1993)
Inhibition of oral carcinogenesis by roasted beans and
roasted coffee bean fractions. In 15th International
Scientific Colloquium on Coffee. pp. 420-425.
Association Scientifique International du Caf6, Paris.
Miller E. G., McWhorter K., Rivera-Hidalgo F., Wright
J. M., Hirsbrunner P. and Sunahara G. I. (1991)
Kahweol and cafestol: inhibitors of hamster buccal
pouch carcinogenesis. Nutrition and Cancer 15, 41-46.
Pietinen P., Aro A., Tuomitehto J., Uusitalo U. and
Korhonen H. (1990) Consumption of boiled coffee is
correlated with serum cholesterol in Finland.
International Journal of Epidemiology 19, 586-590.
Ratnayake W. M. N., Hollywood R., O'Grady E. and
Stavric B. (1993) Lipid content and composition of coffee brews prepared by different methods. Food and
Chemical Toxicology 31, 263-269.
Ratnayake W. M. N., Pelletier G., Hollywood R.,
Malcolm S. and Stavric B. (1995) Investigation of the
coffee lipids on serum cholesterol in hamsters. Food and
Chemical Toxicology 33, 195-201.
Sanders T. A. B. and Sandaradura S. (1992) The cholesterol-raising effect of coffee in the Syrian hamster.
British Journal of Nutrition 68, 431434.
Sanguigni V., Gallu M. P., Rul~ni M. P. and Strano
A. (1995) Effects of coffee on serum cholesterol and
lipoproteins: the Italian brewing method. European
Journal of Epidemiology 11, 75-78.
Stensvold I., Tverdal A. and Foss O. P. (1989) The effect
of coffee on blood lipids and blood pressure. Results
from a Norwegian cross-sectional study, men and
women, 40-42 years. Journal of Clinical Epidemiology
42, 877-884.
Thelle D. S.~ Arnesen E. and Forde O. H. (1983) The
Tromso heart study: does coffee raise serum cholesterol?
New England Journal of Medicine 308, 1454-1457.
Thelle D. S., Heyden S. and Fodor J. G. (1987) Coffee
and cholesterol in epidemiological and experimental studies. Atherosclerosis 67, 97 103.
Thelle D.S. and Van der Stegen G. (1990) In Coffee and
Coronary Heart Disease, Special Emphasis on the CoffeeBlood Lipids Relationship. Edited by D.S. Thelle and G.
Van der Stegen. pp. 9-15. Gotab, Stockholm.
Urgert R., Schulz A. G. M. and Katan M. B. (1995a)
Effects of cafestol and kahweol from coffee grounds on
serum lipids and serum liver enzymes in humans.
American Journal of Clinical Nutrition 61, 149-154.
Urgert R., van der Weg G., Kosmeijer-Schuil T. G., van
de Bovenkamp P., Hovenier R. and Katan M. B. (1995b)
Levels of the cholesterol-elevating diterpenes cafestol
and kahweol in various coffee brews. Journal of
Agricultural and Food Chemistry 43, 2167-2172.
Van Dusseldorp M., Katan M. B., Van Vliet T.,
Demacker P. N. M. and Stalenhoef A. F. H. (1991)
Cholesterol-raising factor from boiled coffee does not
pass a paper filter. Arteriosclerosis and Thrombosis 11,
586 593.
Wattenberg L. W. (1983) Inhibition of neoplasia by minor
dietary constituents. Cancer Research 43, 2448 2453.
Wattenberg L. W., Hanley A. B., Barany G., Sparnins
V. L., Lam L. K. T. and Fenwick G. R. (1985)
Inhibition of carcinogenesis by some minor dietary constituents. International Symposium. Princess Takamatsu
Cancer Research Fund 16, 193-203.
Weusten-Van der Wouw M. P. M. E., Katan M. B., Viani
R., Huggett A. C., Liardon R., Lund-Larsen P. G.,
Thelle D. S., Ahola I., Aro A., Meyboom S. and
Beynen A. C. (1994) Identity of the cholesterol-raising
factor from boiled coffee and its effect on liver function
enzymes. Journal of Lipid Research 35, 721-733.