Coffee consumption and CYP1A2 genotype in relation to bone mineral density of the proximal femur in elderly men and women: a cohort study
Nutrition & Metabolism
Coffee consumption and CYP1A2 genotype in relation to bone mineral density of the proximal femur in elderly men and women: a cohort study
Helena Hallstrm 0 3
Hkan Melhus 1 2
Anders Glynn 0
Lars Lind 5
Ann-Christine Syvnen 4
Karl Michalsson 2 3
0 Research and Development Department, Toxicology Division, National Food Administration , Box 622, SE-751 26 Uppsala , Sweden
1 Department of Medical Sciences, Section of Clinical Pharmacology, Uppsala University , SE-751 85 Uppsala , Sweden
2 Uppsala Clinical Research Center (UCR), University Hospital , SE-751 85 Uppsala , Sweden
3 Department of Surgical Sciences, Section of Orthopedics, Uppsala University , SE-751 85 Uppsala , Sweden
4 Department of Medical Sciences, Molecular Medicine, Uppsala University , SE-751 85 Uppsala , Sweden
5 Department of Medical Sciences, Section of Acute and Internal Medicine, Uppsala University , SE-751 85 Uppsala , Sweden
Background: Drinking coffee has been linked to reduced calcium conservation, but it is less clear whether it leads to sustained bone mineral loss and if individual predisposition for caffeine metabolism might be important in this context. Therefore, the relation between consumption of coffee and bone mineral density (BMD) at the proximal femur in men and women was studied, taking into account, for the first time, genotypes for cytochrome P450 1A2 (CYP1A2) associated with metabolism of caffeine. Methods: Dietary intakes of 359 men and 358 women (aged 72 years), participants of the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS), were assessed by a 7-day food diary. Two years later, BMD for total proximal femur, femoral neck and trochanteric regions of the proximal femur were measured by Dual-energy X-ray absorptiometry (DXA). Genotypes of CYP1A2 were determined. Adjusted means of BMD for each category of coffee consumption were calculated. Results: Men consuming 4 cups of coffee or more per day had 4% lower BMD at the proximal femur (p = 0.04) compared with low or non-consumers of coffee. This difference was not observed in women. In high consumers of coffee, those with rapid metabolism of caffeine (C/C genotype) had lower BMD at the femoral neck (p = 0.01) and at the trochanter (p = 0.03) than slow metabolizers (T/T and C/T genotypes). Calcium intake did not modify the relation between coffee and BMD. Conclusion: High consumption of coffee seems to contribute to a reduction in BMD of the proximal femur in elderly men, but not in women. BMD was lower in high consumers of coffee with rapid metabolism of caffeine, suggesting that rapid metabolizers of caffeine may constitute a risk group for bone loss induced by coffee.
Caffeine is the most widely used central nervous system
stimulant in the world. There are several conceivable
health benefits with the intake of caffeine-containing
beverages but they can also produce unwanted health
consequences. Caffeine increases calcium excretion [1-4]
and decreases intestinal calcium absorption , with 5
mg net loss of calcium per cup of coffee . A high
intake of coffee could therefore also induce loss of bone
Results from epidemiological studies investigating the
relation between coffee consumption and bone mineral
density (BMD) in both women [6-14] and men
[12,15-21] have been conflicting, which might be
explained by differences in sample size, method of data
collection and amount of coffee consumed. In addition,
it has been suggested that a high caffeine intake is only
deleterious for bone health when calcium intake is low
. In Sweden, consumption of coffee and thus
caffeine intake is high in a substantial proportion of the
population, making this setting suitable to study the
relation between coffee and BMD and subsequently the
risk of osteoporosis.
Several enzymes are involved in the metabolism of
caffeine, but the most important is cytochrome P450
1A2 (CYP1A2) . The first step in this metabolic
pathway is a N3-demethylation, which results in the
formation of 1,7-dimethylxanthine, i.e., paraxanthine .
A wide variability in CYP1A2 activity between
individuals has been observed . Depending on genotype,
some individuals are regarded as slow metabolizers of
caffeine, while some are regarded as rapid metabolizers
[25,26]. There is also a gender difference with men, on
average, having higher CYP1A2 activity . To our
knowledge, no study has yet investigated how coffee
consumption could affect BMD in relation to the rate of
caffeine metabolism determined by the genetic
constitution of the individuals consuming coffee. However, in a
study of coffee intake, CYP1A2 genotype and risk of
myocardial infarction coffee was associated with an
increased risk of nonfatal myocardial infarction only in
participants regarded as slow metabolizers of caffeine
. Accordingly, in the present study we hypothesize
that the participants genotype for cytochrome CYP1A2
could modify the relation between coffee consumption
and BMD. This is because caffeine exposure of the body
will last for longer periods in the slow caffeine
metabolizers than in the rapid caffeine metabolizers. Until
now, however, the possibility of modulation by genotype
for CYP1A2 has not been considered in studies of coffee
consumption and BMD.
The principal aim of this study was to investigate the
relation between consumption of coffee and BMD of the
proximal femur in a population-based cohort of
70year-old Swedish men and women. A secondary aim
was to study whether the relation between consumption
of coffee and BMD in the cohort was modified by the
participants genotype for cytochrome P450 1A2
Materials and methods
The Prospective Investigation of the Vasculature in
Uppsala Seniors (PIVUS)  has been described
previously . In brief, all 70-year-old individuals residing
in Uppsala, Sweden, in 2001-2004 were eligible. Of
these individuals, 2,025 were randomly selected and
invited to participate within 2 months of their 70th
birthday from April 2001 to June 2004. Of those invited,
1,016 (50%) eventually participated in the study. The
participants were examined by measurements of blood
pressure and anthropometry, blood sampling after an
overnight fast, routine medical history and assessment
of BMD using Dual-energy X-ray absorptiometry (DXA)
as described below. The study was approved by the
Ethics Committee of Uppsala University and all
participants gave their written informed consent.
Dietary habits were registered in 850 (84%) of the
participants. Each participant recorded his or her food
consumption during 7 consecutive days using a pre-coded
food diary after instructions from a dietician. The
precoded food diary had been prepared and previously used
by the Swedish National Food Administration (NFA)
and Statistics Sweden in a food survey of 3,000
households in 1989 . The questionnaire has been validated
[30,31]. The menu book included written instructions
with an example on how to complete the book. The
record sheets started with day 1 followed by six
additional days. For each meal (breakfast, lunch, dinner and
snacks), the respondent was asked to specify where and
at what time the meal was eaten. The amounts
consumed were reported in household measurements or
specified as portion sizes according to a photograph
showing four portion sizes. Coffee and tea consumption
was registered six times daily (breakfast, lunch, supper,
between meals and in the evening).
The daily intake of energy, caffeine, alcohol and
selected nutrients including calcium, vitamin D and A,
was calculated using a computerized program and
information about energy and nutrient contents of foods
from a database from the National Food Administration
that included 1,500 food items, drinks and recipes.
Filtered or brewed coffee is the most popular type of
coffee in the Nordic countries, while it should be noted
that decaffeinated coffee and tea are not typically
consumed in the Swedish diet . One cup of filtered
coffee (150 mL) was estimated to contain approximately
100 mg caffeine . One cup of tea (200 mL) was
estimated to contain about 50 mg of caffeine . No
analyses of caffeine content of the consumed coffee and tea
Measurement of bone mineral density at the proximal
On average, 2 years after the baseline investigation, 898
of 1,016 cohort members agreed to undergo
measurements of BMD (g/cm2) for total proximal femur,
femoral neck and trochanteric regions of the proximal
femur by DXA (DPX Prodigy, Lunar corp., Madison,
WI, USA). This is the site of the most serious
consequences of osteoporosis - the hip fracture , which
constitutes two main fracture categories: the femoral
neck and the trochanteric femoral fracture. When
applicable, both extremities were used in the calculation.
By triple measurements in 15 participants, the precision
error of the DXA measurements of total proximal femur
in our laboratory has been calculated to be about 0.7%.
Genotyping of CYP1A2
A common polymorphism in both Caucasians and
Asians is the variation of the nucleotide at position -163
in intron 1 of the CYP1A2 gene. The C allele at position
-163 in the CYP1A2 gene is considered to confer
decreased inducibility to the enzyme [24,33,34].
Consequently, carriers of a C allele at this position are
regarded as slow metabolizers of caffeine [24,33,34].
Enzyme inducibility is increased by a substitution of C
with A at position -163 in the CYP1A2 gene and
homozygote carriers of the mutated allele are considered
rapid caffeine metabolizers of caffeine. In a previous
study, polymorphisms of rs762551 in the CYP1A2 gene
have been shown to influence the association between
coffee intake and myocardial infarction . However,
because this single nucleotide polymorphism (SNP) was
later not genotyped in HapMap , we chose another
SNP in HapMap, rs11854147, which is in linkage
disequilibrium with rs762551 (R2 = 0.886). The SNP
rs11854147 was genotyped at the SNP Technology
Platform at Uppsala University, Sweden  using the
Illumina BeadStation 500GX and the 384-plex Illumina
Golden Gate assay (Illumina Inc., San Diego, CA, USA)
. The sample success rate was 98.8% and the
reproducibility 100% according to duplicate analysis of 2.4%
of the genotypes. The genotype distribution was in
We had the possibility to include 717 genotyped
participants with both dietary assessment and BMD
measurement in our analysis. All statistical calculations were
performed using SAS (SAS 9.1; SAS Institute Inc., Cary,
NC). The relation between coffee consumption as a
continuous variable and BMD was primarily analyzed by
ordinary linear regression models. We further
categorized coffee intake by quartiles (0-2 cups/day, 3 cups/day,
4 cups/day and more than 4 cups/day), and the least
square means of BMD for each quartile was estimated
on the basis of the regression estimates using the
General Linear Model (GLM). All estimates were
ageadjusted (at time of the DXA measurement) or adjusted
by a multivariable model. The multivariable model
included age, height, weight, total caloric intake; intakes
of vitamin D, vitamin A, calcium, alcohol and tea (all
continuous). Categorized variables included in the
model were smoking (never, current, former) and levels
of leisure physical activity (low, medium, high). Physical
activity was divided into light and hard exercise and
classified as number of activities for at least 30 min per
week. The participant were asked how many times per
week he/she performed light (e.g. walking, gardening)
respectively hard exercise (e.g. running, swimming) for
at least 30 min . Based on the responses to these
questions, three physical activity categories were
constructed: low, medium, and high. The questions used in
PIVUS were similar to the questions used in the
Uppsala Longitudinal Study of Adult Men (ULSAM)
cohort . The questions in ULSAM have been
Our hypothesis that the participants CYP1A2 genotype
could potentially modify the relation between coffee
intake and BMD was tested in high consumers (both
sexes) of coffee (4 cups or more per day). Slow
metabolizers were defined as participants with genotypes C/T
(40.6%) or T/T (10.7%) while rapid metabolizers were
those with genotype C/C (48.7%). Average
multivariableadjusted BMD values of slow and rapid metabolizers
were compared. To eliminate potential inducing effects
of smoking on CYP1A2 the analyses were repeated in
nonsmoking participants only.
We additionally analyzed whether there existed a
difference in adjusted mean BMD values in men and
women with a high consumption of coffee (4 cups or
more per day) according to their calcium intake: low
(<600 mg/day), intermediate (600-1200 mg/day) and
high (>1200 mg/day) total calcium intake (including diet
Characteristics of the participants in relation to
consumption of coffee are displayed in Table 1. Half of the
participants reported consumption of 3 or 4 cups of
coffee daily and one fourth reported an intake of more than
4 cups of coffee per day. High consumers of coffee in
both men and women had a higher intake of energy and
nutrients. However, their body mass index (BMI) was
similar to that for low consumers of coffee. Self-reported
leisure physical activity was also comparable between
categories of coffee consumers, whereas current
smoking was more prevalent in both men and women with
high consumption of coffee compared with those who
drank none or small amounts (0-2 cups) of coffee.
After multivariable adjustment, there was a trend of
decreased BMD at the total proximal femur with
increasing amounts of coffee consumed (p for trend
0.04) (Table 2). Men who consumed 4 cups of coffee or
more per day had a 4% lower BMD at the total proximal
femur as compared with men who drank 0-2 cups per
day (p = 0.04). This difference was not observed in the
female participants. BMD of the femoral neck and
trochanteric region of the proximal femur was reduced by
3-5% in men consuming 4 cups or more of coffee per
day (femoral neck p = 0.05 and trochanter region p =
0.01 - data not shown). Results for caffeine intake
mirrored those for coffee consumption (data not shown).
Multivariable-adjusted mean values in rapid and slow
metabolizers (men and women) of coffee with a coffee
consumption of 4 cups or more per day are displayed in
Figure 1. Lower (approximately 2-4%) BMD values were
found in rapid metabolizers of caffeine. The differences
reached statistical significance at the femoral neck (p =
0.01) and trochanter region (p = 0.03), but not at the
total proximal femur (p = 0.10) (Figure 1). Because
Table 1 Baseline characteristics of the participants by amount of coffee consumption at the 1st investigation of the
Men (n = 359)
Categories of coffee consumption (cupsb/day)
Women (n = 358)
Categories of coffee consumption (cups/day)
a All values are mean SD (continuous variables) or frequencies (categorical variables). Values in parentheses are frequencies expressed in percent. b The volume
of one cup of coffee is 150 mL
smoking is known to induce CYP1A2, current smokers
(n = 69) were excluded in an extended analysis of the
cohort. The results of this analysis remained essentially
unchanged in terms of effects upon BMD (data not
shown). There were, furthermore, no statistical
differences between slow and rapid metabolizers with a low
consumption of coffee or between rapid metabolizers
with a low consumption of coffee and slow metabolizers
with a high consumption (data not shown).
High consumers (men and women) of coffee (4 cups
or more per day) with a high calcium intake (more than
1200 mg per day) did not have higher adjusted average
BMD values compared with those with high coffee
consumption and low (<600 mg/day) or intermediate
(6001200 mg/day) calcium intake (Data not shown). Finally,
tea was consumed by 439 participants (about 60%) in
the study. Tea consumption was not associated with
multivariable-adjusted BMD (parameter estimate per
cup of tea -0.0014 (95% confidence interval -0.0106,
0.0078; p = .77).
In this cohort the consumption of coffee was high. We
observed a decrease in BMD of the proximal femur in
men consuming 4 cups of coffee or more daily. In high
consumers of coffee, rapid metabolizers had lower BMD
values than slow metabolizers of caffeine. A potential
risk group more prone to develop osteoporosis might,
thus, have been identified.
The observed decrease in BMD in male high
consumers of coffee could be estimated to correspond to an
approximately 30% increased risk of hip fracture, which
would imply a considerable increase in view of public
health . This increased risk might have impact on
total osteoporotic fracture health economy. This is
illustrated by the fact that the number of hip fractures
worldwide in the year of 2000 was estimated to 1.6
million . The global cost for hip fractures is rising and
by 2050 it has been estimated to be about 132 billion
US dollars .
Earlier studies in men [12,15-21] have not observed
any statistically significant relation between
consumption of coffee and BMD. It should be noted, however,
that some of the studies were small [12,15,21]. In most
studies [17-21] the exposure was defined as caffeine
intake from both coffee and tea. This approach may not
be optimal because both beverages contain several other
bioactive substances that may modify the effects of
caffeine. Furthermore, many of the earlier studies do not
clearly state the exposure as amount of coffee or
caffeine consumed [15,17,18,20]. When stated, the average
intake of coffee/caffeine varied from approximately 200
mg caffeine per day [19,21] or less than two cups of
coffee per day  to 3 cups per day in one study .
Table 2 Age-adjusted and multivariable-adjusteda bone mineral density (BMD) in the proximal femur (mean and 95%
CI) of the PIVUS cohort by amount of coffee consumption
Categories of coffee consumption (cupsb/day)
All participants (n = 717)
p = 0.15
p = 0.10
p = 0.91
Men (n = 359)
Women (n = 358)
p = 0.09
p = 0.04
p = 0.73
p = 0.08
p = 0.04
p = 0.62
a Adjusted by age at the BMD-measurement, height, weight, total caloric intake, vitamin D intake, vitamin A intake, calcium intake, alcohol intake, intake of tea
(all continuous), smoking (never, current, former) and levels of leisure physical activity (low, medium, high) b The volume of one cup of coffee is 150 mLc Per cup
Figure 1 Mean adjusted BMD (bone mineral density) of the total proximal femur in men and women with a high consumption of
coffee (4 cups or more per day) by CYP1A2 polymorphism. The error bars indicate 95% confidence intervals (CI) and the p-values refer to
comparisons between slow and rapid metabolizers at each site. Mean values are adjusted by age at the BMD measurement, height, weight, total
caloric intake, vitamin D intake, vitamin A intake, calcium intake, alcohol intake, intake of tea (all continuous), smoking (never, current, former)
and levels of leisure physical activity (low, medium, high).
In our study, compared to most other studies, mean
intake of caffeine and consumption of coffee was higher:
367 mg/day and 3.2 cups/day, respectively.
In the majority of the studies of women no relation
between consumption of coffee or intake of caffeine and
BMD has been detected [6,7,13,14]. Nevertheless, a
weak negative relation between coffee or caffeine and
BMD has been observed [9,11,22,44-48] but the relation
between BMD and intake of coffee/caffeine has been
attenuated by adequate intake of milk/calcium [9,22,47].
Our results do not support these latter findings but few
of our participants had a low calcium intake. In general,
the studies in women thus provided limited evidence for
the existence of a relation between intake of
coffee/caffeine and effects on BMD, which is accordance with our
results. It should be noted that as in the studies of men,
many of the studies in women were small [9,49-57]. In
addition, no separate analyses of coffee and tea were
carried out in the majority of studies of women
[8-10,44,45,48-56,58,59]. Average intake of coffee or
caffeine seems to have been low or modest in some studies
[10,44,53,55,56], i.e., lower than in the present
There is evidence for females having lower activity of
CYP1A2 than men . With a higher CYP1A2 activity
in men, caffeine will be more rapidly metabolized and
the concentrations of metabolites like paraxanthine will
become higher in relation to the concentration of
caffeine. The deleterious effect of coffee consumption on
bone may be an effect of caffeine metabolites.
Consistent with this theory is that we observed lower BMD
among rapid compared to slow metabolizers of caffeine
with a high coffee consumption. Moreover, we found
lower BMD among male high consumers of coffee but
not among such women, an observation that may be
explained by higher CYP1A2 activity in men . In
addition, we did not find any statistical differences
between slow and rapid metabolizers with a low
consumption of coffee or between slow metabolizers with a
high consumption of coffee and rapid metabolizers with
a low consumption. Our results may thus indicate that a
certain level of metabolites must be reached in order to
observe a negative effect on BMD. There are, however,
no published data regarding effects of metabolites of
caffeine on BMD. Therefore more studies are clearly
warranted in order to investigate possible mechanisms
of interactions regarding caffeine intake and CYP1A2
genotype in relation to BMD. How caffeine or its
metabolites exert effects on bone can theoretically be
explained by other mechanisms than by reduced renal
calcium conservation. According to some in vitro
studies, caffeine may interfere with bone remodelling
process. Tsuang et al (2006)  suggested that caffeine
may have deleterious effect on the viability of rat
osteoblasts, which could enhance the rate of osteoblast
apoptosis. In addition, Lu et al (2008)  has
demonstrated that cell viability also decreased in human
osteoblasts treated with caffeine in a dose-dependent manner
mainly due to apoptosis. Zhou et al (2009) 
hypothesise, however, that bone marrow-derived mesenchymal
stem cells, which are precursor cells of osteoblasts, may
be the real target cells of caffeine-induced osteoporosis
in vivo. However, it remains to be demonstrated
whether a mechanism including direct effects of caffeine
or its metabolites on cells involved in the remodelling
process could be of importance also in vivo at dosages
of relevance to humans.
It has been demonstrated that both the parent
compound, caffeine, as well as paraxanthine, might be
teratogenic after administration of very high doses in mice
with skeletal malformations as a consequence .
Caffeine is cleared more quickly than paraxanthine and 8
hours after caffeine intake, plasma concentrations of
paraxanthine levels exceed those of caffeine . With
long-term exposure of high doses of caffeine there is
substantial accumulation of paraxanthine [65,66].
Paraxanthine has in vitro been found to be a potent
suppressor of transforming growth factor beta (TGF-b) ,
which stimulates bone formation, and TGF-b deficiency
may result in osteoporosis . Interestingly,
paraxanthine has been found to be the most powerful
pharmacological repressor of hepatocellular TGF-b
dependent connective tissue growth factor expression
among the drug family of methylxanthines, including
caffeine . The major caffeine derivatives, including
paraxanthine, have common mechanisms of action, i.e.
competitive antagonism of the adenosine interaction
with A1 and A2 receptors. Deactivation of the adenosine
receptors, which are expressed in bone cells, can result
in reduced bone formation .
Advantages and limitations of our study
To our knowledge, this is one of few population-based
studies investigating possible effects of coffee and tea
consumption on BMD in both men and women. In
contrast to most other studies, the majority of the
participants in our study consumed high amounts of coffee.
We had a sufficient number of participants to detect
even modest associations. An additional strength is that
we did not focus on caffeine intake but on the exposure
of coffee and tea separately. This distinction may be
important because some studies have indicated that
consumption of tea could have a positive influence on
BMD, which could counteract the negative influence of
coffee. Tea consumption in our study was low and
adjusted for in the statistical analyses. The possible
modification by genotype for CYP1A2 inducibility has
not previously been investigated. We also had the
possibility to consider several conceivable covariates in
the analysis, including nutrients, physical activity
behavior and smoking.
This study nevertheless has several potential
limitations. In this study we have measured BMD in the
proximal femur only. We refrained from including BMD
measurements of the spine since spondylosis is common
in elderly individuals, and this condition can confound
the relative weak association between BMD and coffee as
well as the comparison between sexes. As the
measurement of BMD was on average performed 2 years after the
dietary investigation, the follow-up time was limited.
However, the optimal time between measurements of
coffee consumption and BMD is currently not known.
Nevertheless, it should be noted that earlier studies on
skeletal effects by an exposure that affects calcium
metabolism indicate a lag period of 2-3 years before a steady
state of bone turnover and BMD is reached [70,71].
Statistically significant differences in BMD between
high consumers who were rapid metabolizers and those
who were slow metabolizers of caffeine were generally
confined to the whole study group of both men and
women, probably because statistical power was too low
to attain statistical significance in the groups of each
gender. There were, however, clear tendencies of a
lower BMD in high consuming males who were rapid
metabolizers. In women the same pattern could also be
Because the exposure measurement was based on a
single dietary measurement, there may be some degree
of error in the measurement. The 7-day dietary
recording used in the present study has been found to be valid
 and a high reliability of self-reported measures of
caffeine consumption has previously been shown .
Nonetheless, because no direct measurements of the
caffeine content in the consumed coffee and tea were
performed, we lack data on the actual intake of caffeine.
Still, recall errors of the exposure are known to lead to
conservatively biased estimates.
Temporal changes in the consumption pattern of
coffee and tea by time might be of importance but this was
not assessed since we only determined baseline
frequencies of consumption. However, in the Swedish
Mammography Cohort  it was found that the consumption
of coffee among elderly women during 10 years  was
relatively constant (Personal communication with Dr SC
Larsson, Division of Nutritional Epidemiology, The
National Institute of Environmental Medicine,
Karolinska Institute, Stockholm, Sweden)
A high consumption of coffee (i.e. 4 cups or more per
day) could contribute to a reduction in BMD of the
proximal femur in elderly men. BMD was lower in high
consumers of coffee with rapid metabolism of caffeine,
suggesting that this group of coffee consumers might be
at special risk of bone loss.
We thank Tomas Axelsson, Torbjrn st and Marie Lindersson at the SNP
Technology Platform in Uppsala, Sweden for assistance with the genotyping.
This study was supported by grants from the Swedish Research Council. The
SNP genotyping was supported by the Knut and Alice Wallenberg
foundation and Uppsala University.
The authors contributions were as follows - HH and KM: designed the study;
HH: analyzed the data and drafted the manuscript; LL: recruited the
participants, obtained funding, collected data and is principal investigator for
the cohort; A-CS: organized the genotyping; HM and AG contributed to the
study design and performance, and assisted with the editing of the
manuscript and KM revised the manuscript, supervised the study, collected
data and obtained funding. All authors have read and approved the final
The authors declare that they have no competing interests.
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