Prostaglandins, Leukotrienes and Essential Fatty Acids 109 (2016) 812

Contents lists available at ScienceDirect

Prostaglandins, Leukotrienes and Essential

Fatty Acids
journal homepage: www.elsevier.com/locate/plefa

Is vaccenic acid (18:1t n-7) associated with an increased incidence

of hip fracture? An explanation for the calcium paradox
Kei Hamazaki a,n, Nobuo Suzuki b, Kei-ichiro Kitamura c, Atsuhiko Hattori d,
Tetsuro Nagasawa e,1, Miho Itomura e,f, Tomohito Hamazaki e,f
a

Department of Public Health, Faculty of Medicine, University of Toyama, 2630 Sugitani, Toyama city, Toyama 930-0194, Japan
Noto Marine Laboratory, Division of Marine Environmental Studies, Institute of Nature and Environmental Technology, Kanazawa University, Noto-cho,
Ishikawa 927-0553, Japan
c
Faculty of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa city, Ishikawa 920-0942, Japan
d
Department of Biology, College of Liberal Arts and Sciences, Tokyo Medical and Dental University, Ichikawa, Chiba 272-0827, Japan
e
Division of Clinical Application, Department of Clinical Sciences, Institute of Natural Medicine, University of Toyama, Toyama city, Toyama 930-0194, Japan 2
f
Department of Internal Medicine, Toyama Jonan Onsen Daini Hospital, Toyama city, Toyama 933-8271, Japan
b

art ic l e i nf o
Article history:
Received 27 January 2016
Received in revised form
4 April 2016
Accepted 4 April 2016
Keywords:
Calcium paradox
Dairy products
Goldsh scale
Hip fracture incidence
Milk
Osteoblasts
Osteocalcin
trans Fatty acid
Collagen type I

a b s t r a c t
High calcium intake may increase hip fracture (HF) incidence. This phenomenon, known as the calcium
paradox, might be explained by vaccenic acid (18:1t n-7, VA), the highly specic trans fatty acid (TFA) present
in dairy products. First, we ecologically investigated the relationship between 18:1 TFA intake and HF incidence using data from 12 to 13 European countries collected before 2000; then we measured the effects of
VA and elaidic acid (18:1t n-9, EA) on osteoblasts from goldsh scales (tissues very similar to mammalian
bone), with alkaline phosphatase as a marker; and nally we measured the effect of VA on mRNA expression
in the scales for the major bone proteins type I collagen and osteocalcin. HF incidence was signicantly
correlated with 18:1 TFA intake in men (r0.57) and women (r0.65). Incubation with 1 mol/L VA and EA
for 48 h signicantly decreased alkaline phosphatase activity by 25% and 21%, respectively. Incubation of
scales with 10 mol/L VA for 48 h signicantly decreased mRNA expression for type I collagen and osteocalcin (by about 50%). In conclusion, VA may be causatively related to HF and could explain the calcium
paradox. It may be prudent to reduce 18:1 TFA intake, irrespective of trans positions, to prevent HF.
& 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Consumption of dairy products in Japan is much lower than in
Western countries, with Japanese adults consuming, on average,
only about 500 mg of calcium per day [1]. However, the incidence of
hip fracture (HF), an osteoporosis-related fracture, is lower in Japan
than in Western counties [2]. Using data from several countries,
Hegsted was the rst to show that HF incidence in women was
positively associated with per capita calcium consumptionthe socalled calcium paradoxand also per capita protein consumption
[3]. Abelow et al. later found very similar associations in a larger
number of countries [4]. Furthermore, diets rich in animal protein
and sodium have been shown to increase calcium loss. A recent
meta-analysis of 30 studies comparing the effects of high and low
n

Corresponding author.
E-mail address: keihama@med.u-toyama.ac.jp (K. Hamazaki).
1
T.N. is currently a free scientist.
2
This division was integrated in 2012 and no longer exists.

http://dx.doi.org/10.1016/j.plefa.2016.04.001
0952-3278/& 2016 Elsevier Ltd. All rights reserved.

protein diets on renal function showed that high protein diets resulted in signicantly more urinary calcium excretion (mean difference: 25 mg/day, 95% condence interval 1437, Po0.001) than
normal or low protein diets [5]. This small difference in urinary
calcium excretion of 25 mg/day is not likely clinically relevant.
Recently, a study in central Sweden by Michalsson et al. found
that high consumption of milk was signicantly associated with high
all-cause mortality in both sexes and with HF incidence in women
but not in men; the Swedish Mammography Cohort involved 61,433
women (baseline: 19871990) and the Cohort of Swedish Men involved 45,339 men (baseline: 1997) [6]. Michalsson et al. suspected
the lack of benecial effects of milk was due to the presence of Dgalactose, the main source of which is milk, where it is contained in
the form of lactose. They suggested that D-galactose was a causative
factor given that women with a high intake of cheese or fermented
milk products, which contain little D-galactose, had 1015% lower
mortality and HF rates than women with low intake, and given the
results of animal experiments [7,8].
Although D-galactose may be an important explanatory factor

K. Hamazaki et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 109 (2016) 812

for the calcium paradox, it was not correlated with HF in men [6].
This means another dairy product-specic factor could cause HF.
One candidate is vaccenic acid (VA, 18:1t n-7), the major trans fatty
acid (TFA) in dairy products and not abundantly found in partially
hydrogenated oil [9]. We, therefore, became interested in investigating the ecological relationship with and biological effects
of VA as another major potential explanatory factor for HF.

2. Methods
2.1. Ecological study
We rst investigated whether 18:1 TFA consumption was associated with HF by using ecological data from Western European
countries. Here, such consumption largely represented the

Men
r=0.57, p<0.05 (without Japan)

Hip fracture incidence at age 80 years

(1/100,000)

Women
r=0.65, p=0.02 (without Japan)
(1/100,000)

1,000

NOR ICE

NOR

SWE

SWE
DEN

1,600
800

DEN
ICE
FIN

GRE

GER

600

ITA

GER
POR

800

SPA

GRE
SPA

POR

ITA

JPN
JPN

200

NET

FIN

NET

UKI

400

UKI

1,200

FRA

400

FRA

0
0.75 1.0

1.5 2.0

3.0

1.0

4.0

1.5

2.0

3.0

18:1 TFA intake (g/day, log scale)

Hip fracture incidence at age 80 years

Men
r=-0.22, p=0.48 (without Japan)
(1/100,000)
1,000

Women
r=-0.46, p=0.11 (without Japan)
(1/100,000)

NOR

NOR

SWE

1,600

ICE
SWE
DEN

DEN

800

ICE

FIN

600
GER

UKI

400

UKI
NET
FIN

NET
SPA

POR
JPN

GER

1,200

GRE

POR

800

SPA

JPN

ITA

FRA

ITA GRE

FRA

400

200

0
15

20

30

40

50

10

15

20

30

40

Oleic acid (18:1 cis) intake (g/day, log scale)

Fig. 1. Association between 18:1 trans fatty acid and oleic acid intake, and hip fracture incidence at age 80 years. Values for 18:1 trans fatty acid and oleic acid intake were
available only for men in Italy and Portugal. Women's values for those two countries () were estimated from the mean ratios between men and women in countries where
both values were available (nine countries). Values for sex-specic intake were not available in the United Kingdom and Spain (), and only both sex-combined data were
available. Sex-specic intakes were estimated from the abovementioned ratios and geometric means of both sexes. (See Section 2 for details.) The r values for men and
women were r 0.57, Po 0.05 and r 0.65, P 0.02, respectively (both r values were calculated without Japan ()).

10

K. Hamazaki et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 109 (2016) 812

combined value of VA (the major TFA in dairy products) and

elaidic acid (EA, 18:1t n-9, the trans isomer of oleic acid, most
abundant in partially hydrogenated oils). We considered these
countries to offer the best data for comparing TFA consumption
and incident HF because the following confounding factors are
more homogeneous among these countries than in any other part
of the world: food intake, income, social security, public health
service, culture, etc. Because consumption of TFAs has been decreasing in recent years, we collected rather old datafrom the
TRANSFAIR Study [10]when restriction of TFAs was not that
stringent. The TRANSFAIR Study collected food samples from 14
Western European countries between June 1995 and April 1996,
using a market basket method. This method involved purchasing a
maximum of 100 foods representative of the total fat intake from
regular commercial outlets for consumer products (or in some
cases purchased products for industrial use only) and sampling
these foods according to a standardized stepwise approach,
working within budget restrictions [11]. Food samples were similarly processed, kept at 20 C in each country, and transported
to a central laboratory in the Netherlands for TFA measurement
[11]; the latter step is one of the most important techniques for
quality control for international comparisons. Given that the study
was not able to separate individual 18:1 TFA isomers completely,
we also used 18:1 TFA isomers as a group.
Because the HF incidence in women is about two-fold more
than that in men [2], HF investigations must be performed for each
sex. However, four countries in the TRANSFAIR Study did not have
sex-specic data for 18:1 TFA intake but had men's data only (Italy
and Portugal) or those for both sexes combined (Spain and the
United Kingdom). The women's 18:1 TFA values for Italy and
Portugal were estimated by multiplying the corresponding men's
data with the coefcient 0.83, which was the mean ratio between
women's and men's data in the nine countries where sex-specic
data were available. Belgium was not included for the coefcient
calculation because it was not found in the report we referred to
for HF risk by country [2]. Sex-specic values for Spain and the
United Kingdom were estimated by multiplying sex-combined
data with the coefcients 1.10 (1/0.83) for men and 0.91
( 0.83) for women. As for oleic acid (as the control), again four
countries in the study did not have sex-specic data and therefore
we imputed the data in the same manner mentioned above. The
mean ratio of oleic acid between women's and men's data in the
nine countries where sex-specic data were available was 0.74.
Sex-specic values for Spain and the United Kingdom were estimated by multiplying sex-combined data with the coefcients 1.16
( 1/0.74) for men and 0.86 ( 0.74) for women. Japanese data
for total TFA was from [12] and mean proportion of 18:1 TFA to
total TFA was from [10], and are shown in Fig. 1 just for reference.
Japanese data for oleic acid was from [13].
We selected data on HF risk by country from a report by Kanis
et al. [2] They summarized HF incidence for 29 countries by reviewing published sources, and covered 13 of the 14 countries that
the TRANSFAIR Study investigated [10]. The documents used for
the HF incidence estimation in those 13 countries were mostly
published in the 1990s, with the mean year of publications being
1995 74 [2], very near the food sampling years for the TRANSFAIR
Study (1995 and 1996).

female goldsh (3040 g) were purchased from a commercial source

(Higashikawa Fish Farm, Yamatokoriyama, Nara). Scales were carefully extracted under anesthesia, left to regenerate for 14 days, and
then used as the source of osteoblasts and osteoclasts. Alkaline
phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP)
activities were used as surrogate markers for osteoblast and osteoclast activity, respectively. Activity and sensitivity to hormones were
higher in regenerating scales than mature scales [16]. Regenerating
scales were incubated for 24 or 48 h in Leibovitz's L-15 medium with
antibiotics containing either a fatty acid (Sigma-Aldrich Corporation,
St. Louis, Missouri) or a vehicle alone for controls. Control scales were
sampled from the same part of the contralateral side of the goldsh.
Free fatty acids were dissolved in ethanol and diluted in medium at
0.110 mol/L. After incubation, scales were assayed for ALP and
TRAP activity [16]. All experimental procedures were conducted in
accordance with the Guide for the Care and Use of Laboratory Animals of Kanazawa University.
2.3. Analysis of mRNA expression of type I collagen and osteocalcin
Our analytical methods for real time-PCR are described in a
previous report [17]. The primers used for osteocalcin, type I collagen, and -actin for goldsh scales are described elsewhere [14].
Briey, scales collected from goldsh under anesthesia were incubated for 48 h in L-15 medium supplemented with VA (10 mol/
L) or vehicle. After incubation, the scales were immediately frozen
and kept at  80 C until analysis. Total RNA was prepared from
the scales, and complementary DNA was synthesized with 1 g
RNA. PCR amplication was analyzed by real-time PCR apparatus
(Stratagene). The -actin mRNA level was used as an internal
standard.
2.4. Statistics
The least square method was used to investigate the correlation
between incidence of HF and 18:1 TFA intake. The effects of fatty
acids on ALP and TRAP were compared with control values by the
t-test after ANOVA. In the experiments involving real time-PCR,
the data were analyzed using the paired t-test. Data are expressed
as means 7SEM. Differences were considered signicant at
Po 0.05.

3. Results
Our ecological study revealed that HF incidence was positively
associated with 18:1 TFA consumption but not with oleic acid
(Fig. 1). Experiments with osteoblasts showed that ALP activity, the
marker of osteoblasts, was dose-dependently inhibited after 48 h
incubation with VA and EA but not with oleic acid (18:1c n-9),
which is the cis counterpart of EA (Fig. 2). Similarly, cis vaccenic
acid (18:1c n-7) did not affect such activity (data not shown). In
our experiments with osteoclasts, VA showed no effect on TRAP
activity, the marker of osteoclasts (data not shown). Lastly, the
mRNA expression of osteocalcin and type I collagen after goldsh
scales were incubated for 48 h with 10 mol/L VA revealed around
a 50% reduction in both cases (Fig. 3).

2.2. Experiments with osteoblasts and osteoclasts from goldsh

scales

4. Discussion

Goldsh (Carassius auratus) scale regeneration is known to share

similarities in gene expression and morphology with intramembranous bone regeneration [14]. The effects of free fatty acids
on osteoblasts and osteoclasts in experiments using these goldsh
scales were reported in our previous paper [15]. Briey, mature

In our ecological study we used TFA data from the TRANSFAIR

Study reported in 1999 [10]. We used this particular study's data
because TFA restrictions were not so strict at the time of the study
newer studies would be confounded with wider variations of
TFA intake since in the last decade or so many countries have

K. Hamazaki et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 109 (2016) 812

Vaccenic acid (18:1t n-7)

Elaidic acid (18:1t n-9)

0.5

0.5

0.4

0.4

0.3

**

Oleic acid (18:1c n-9)

0.5
0.4

**

0.3

0.3

0.2

0.2

0.2

0.1

0.1

0.1

0.1

10

11

0.1

10

0.1

10
(mol/L)

Fig. 2. Depressive effects of 18:1t and 18:1c fatty acids on alkaline phosphatase activity in goldsh scales. Regenerating scales were incubated with various concentrations of
indicated trans fatty acid or without them (control) for 48 h. p-Nitrophenyl-phosphate was used as the substrate. #: nmol of p-nitrophenol/min/mm2 of scale. P o 0.1,
*Po 0.05, **P o 0.01. n 8 for each column.

Relative ratio (osteocalcin/-actin)

10
8

*
**

4
2
0

24

48

Relative ratio (type I collagen/-actin)

Control
Vaccenic acid

Osteocalcin

Type I collagen

0.5

0.4
0.3

**
***

0.2
0.1
0

24

48

(h)

Fig. 3. Depressive effects of vaccenic acid on osteocalcin and type I collagen mRNA expressions in goldsh scales. Regenerating scales were incubated with 10 mol/L
vaccenic acid or without trans fatty acids (control) for the hours indicated. Values were normalized to -actin mRNA levels (n 6). *Po 0.05, **Po 0.01, ***Po 0.005.

started paying attention to TFA intake. The use of such data would
also offer a better chance of observing signicant correlations with
the deleterious effects of TFA, if any such effects exist. Another
reason we used the TRANSFAIR Study data was that TFAs were
centrally measured. Without this type of quality control, TFA intake could not be accurately measured because it was not until
2005 that the American Oil Chemists' Society recommended SP2560 columns for TFA measurement [18].

We believe our goldsh scale system has a number of benets.

The effects of bone-affecting hormonesparathyroid hormone
[19], calcitonin [20], drugs (bisphenol A [21]), endocrine disruptors
(tributyltin [22]), and heavy metals [23]on osteoblastic and osteoclastic activities using goldsh scales were exactly the same as
those when using mammalian bone. Moreover, we can incubate
both osteoblasts and osteoclasts in their home positions at the
same time. In this way, interactions between these two cell types

12

K. Hamazaki et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 109 (2016) 812

are preserved. This is a crucial point because Suzuki and Hattori,

two of the present authors, reported that melatonin depressed
both osteoclastic and osteoblastic activities using our scale system
[24], but according to other researchers it stimulated proliferation
and differentiation of isolated (not co-cultured) mammalian osteoblasts [25,26]. In addition, Suzuki et al. previously found that
incubation for 64 h in our scale system with 0.1 mol/L cadmium
and methyl mercury (notorious bone-toxic heavy metals) decreased ALP activity by 27% and 20%, respectively [23]. These rates
are comparable with those in the present study of around 25% and
21% for 48 h incubation with 1 mol/L VA and EA (see Fig. 2).
In order to strengthen our experiments, mRNA expression of
osteocalcin and type I collagen in scales was also analyzed. Expression was signicantly reduced with VA (Fig. 3). Taken together,
these ndings suggest that VA inhibits osteoblast activity at least
in goldsh scales.
Because TFAs and cis-fatty acids differ only in the double-bond
conguration of their molecules, TFAs might exert effects by
competitively inhibiting the metabolism and/or signal transmission with fatty acids that have a cis-double bond(s). However,
incubation with cis-vaccenic and oleic acids did not enhance osteoblast activity. Consequently, the theory of competition between
trans and cis conguration within the fatty acids with the same
length may not hold.
This study has several limitations. First, in general, ecological
studies are useful for generating hypotheses but the ndings for
groups should not be applied to individuals and no denite conclusions can be drawn from such studies. Second, we examined
data from European countries and Japan only, and therefore these
data are not representative of HF patients worldwide. Third, we
were able to use 18:1 TFA isomers only as a group and so were not
able to distinguish the separate effects of EA and VA.
In conclusion, this study is the rst to evaluate the relationship
between 18:1 TFA and HF incidence. The ndings suggest that 18:1
TFA may be an essential factor in explaining the calcium paradox
and that intake of 18:1 TFA may be causatively related to HF. It
may be prudent, therefore, to reduce 18:1 TFA intake, irrespective
of trans positions, to prevent HF. Future research including large
cohort studies is warranted to evaluate the association between
individual 18:1 TFA isomers and HF incidence.

Conict of interest statement

K.H. received research support from an Intramural Research
Grant for Neurological and Psychiatric Disorders from the National
Center of Neurology and Psychiatry and a grant from the Japan
Society for the Promotion of Science; consultant fees from Polyene
Project, Inc.; and scholarship donations from Otsuka Pharmaceutical Co., Ltd. T.H. has received lecture fees from Takeda Pharmaceutical Co., Ltd. and travel expenses from Aker BioMarine. N.S., K.
K., A.H., T.N. and M.I. reported no conicts of interest.

Acknowledgments
This study was supported in part by grants to N.S. (Grant-in-Aid
for Scientic Research [C] No. 24620004 by JSPS), to K.K. (Grant-inAid for Scientic Research [C] No. 15K01705 by JSPS), and to A.H.
(Grant-in-Aid for Scientic Research [C] No. 24570068 by JSPS).
The funding sources had no role in the study design; the collection, analysis, and interpretation of data; writing of the report; or
the decision to submit the paper for publication.

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