0021-972X/01/$03.00/0
The Journal of Clinical Endocrinology & Metabolism
Copyright © 2001 by The Endocrine Society
Vol. 86, No. 3
Printed in U.S.A.
The BB-Paraoxonase Genotype Is Associated with
Impaired Brachial Reactivity after Acute
Hypertriglyceridemia in Healthy Subjects
GIUSEPPE PAOLISSO, DANIELA MANZELLA, MARIA ROSARIA TAGLIAMONTE,
MICHELANGELA BARBIERI, RAFFAELE MARFELLA, GUIDO ZITO,
MASSIMILIANO BONAFÈ, DARIO GIUGLIANO, CLAUDIO FRANCESCHI, AND
MICHELE VARRICCHIO
Department of Geriatric Medicine and Metabolic Diseases, Second University of Naples, I-80138
Naples, Italy; and Department of Experimental Pathology, University of Bologna (M.B., C.F.), I-40100
Bologna, Italy
ABSTRACT
The possible relationship between paraoxonase (PON) gene polymorphism and brachial reactivity in healthy adult subjects in the
presence of acute hypertriglyceridemia (HT), as a prooxidant factor,
was investigated. In 101 healthy subjects the response to flowinduced vasodilatation was measured before and after Intralipid infusion. In the same subjects the A/B PON polymorphism was detected.
The frequency was 0.545 for AA genotype, 0.356 for the AB genotype,
and 0.099 for the BB genotype. At baseline all genotype groups had
a similar increase in brachial artery diameter and flow. After Intralipid infusion, subjects sharing the BB genotype had a significant
decrease vs. baseline values in changes in brachial artery diameter (P
for trend ⬍ 0.001 vs. the other genotypes), but not in flow. In a
P
REVIOUS STUDIES have demonstrated an association
between the common polymorphism at codon 192 in
the human paraoxonase 1 gene (PON1) and the increased
risk for coronary heart disease (1– 4). The relationship between PON1 gene polymorphism and atherosclerosis seems
to be due to a change in antioxidant properties of the high
density lipoprotein (HDL) cholesterol-associated enzyme
paraoxonase (PON) (1). On the other hand, endothelial dysfunction has been suggested as an early marker of atherosclerosis (5–7). In particular, patients with coronary heart
disease (CHD) have an impaired endothelium-dependent
relaxation in both brachial (8) and coronary arteries (9), with
a close correlation between the two sites, thus indicating a
systemic nature of endothelial dysfunction. Thus, brachial
reactivity has been considered a good method for studying
endothelial function in subjects with and without atherosclerosis (10). Interestingly, an acute rise in plasma triglycerides concentrations has been shown to be a prooxidant
factor (11), to provoke endothelial dysfunction (12), and to
impair brachial reactivity (5). Such a finding is in agreement
with the evidence that elevated plasma triglycerides conReceived August 4, 2000. Revision received October 27, 2000. Accepted November 2, 2000.
Address all correspondence and requests for reprints to: Prof. Giuseppe Paolisso, IV Divisione di Medicina Interna e Malattie
dell’Invecchiamento, Department of Geriatric Medicine and Metabolic
Diseases, Second University of Naples, Piazza Miraglia 2, I-80138 Naples, Italy. E-mail: gpaoliss@tin.it.
subgroup of 55 subjects distributed among the 3 PON genotypes the
same study protocol was repeated by buccal nitroglycerine administration to study the endothelium-independent vasodilatation. Again,
subjects with the BB genotype had the worse vasodilation (P for
trend ⬍ 0.001). Furthermore, subjects sharing the BB genotype had
the lowest endothelium-independent and -dependent changes in diameter (P for trend ⬍ 0.001 vs. the other genotypes) independently of
gender ratio, basal plasma triglycerides concentrations, and changes
in plasma triglycerides concentrations. In conclusion, our study demonstrates that transient HT decreases vascular reactivity more in
subjects with the PON BB genotype than in those with the other PON
genotypes. (J Clin Endocrinol Metab 86: 1078 –1082, 2001)
centrations are an independent risk factor for CHD in noninsulin-dependent diabetes mellitus (13). Due to the pivotal
role played by oxidative stress in endothelial dysfunction
and brachial reactivity (14), and as the HDL cholesterolassociated enzyme PON might contribute to the control of
antioxidant activities, it might be postulated that subjects
sharing different PON genotypes might have a different risk
of endothelial dysfunction. Due to the fact that PON gene
polymorphism is associated with increased risk of CHD (2–
4), one can hypothesize this different endothelial behavior to
be the link between PON gene polymorphism and CHD.
Indeed, to the best of our knowledge the association between
PON polymorphism and endothelial dysfunction has not
been investigated. Thus, we aim at investigating the possible
relationship between PON gene polymorphism and brachial
reactivity in healthy adult subjects in the presence of acute
hypertriglyceridema as a prooxidant factor.
Materials and Methods
Study protocol
One hundred and one subjects (45 men and 56 women; mean age,
36.2 ⫾ 14.8 yr) participated after giving informed consent. All individuals were Caucasians and were living in South Italy. Only 34 subjects
were light smokers. None was hypertensive or had clinical signs or
family history of CHD. All subjects had normal glucose tolerance and
were also shown to have normal fasting plasma triglycerides concentrations. All subjects were studied after an overnight fast (at least 12 h).
In all subjects the response to flow-induced vasodilatation was measured twice before and after 1 h after Intralipid (Pharmacia-Upjohn,
1078
PON POLYMORPHISM AND BRACHIAL REACTIVITY
Piscataway, NJ) infusion (0.15 g/kg given as a bolus followed by a
continuous infusion of 0.15 g/kg䡠h until the experiment was finished).
To investigate whether triglyceridemia affects endothelial function in
different PON genotypes in an endothelium-dependent or -independent
manner, flow- and nitroglycerine (NTG)-induced vasodilatation was
studied. For this reason, in a subgroup of 55 subjects distributed among
the 3 different PON genotypes, a second series of experiments was made
with endothelium-independent (NTG-induced) vasodilatation tested
before and after a 1-h infusion of Intralipid. The dose of NTG used was
of 0.4 mg buccal NTG. The study was approved by the ethical committee
of our institution.
Anthropometric determinations
Weight and height were measured using a standard technique. Body
mass index was calculated as body weight (kilograms)/height (meters)2.
Fat-free mass was measured using a four-terminal bioimpedence analyzer (RJL Spectrum Bioelectrical Impedance, IA 101/SC Akern, RJL
System, Florence, Italy).
Brachial reactivity
Brachial reactivity was detected using high frequency ultrasound
technique as reported previously (15). Briefly, all subjects were kept at
rest in the supine position in a temperature-controlled room (22 C) while
heart rate and blood pressure were continuously monitored by a noninvasive technique (Finapress, OMHEDA 2003; Englewood, CO) (16).
The left arm was immobilized in the extended position to allow consistent access to brachial artery for imaging. Brachial artery diameter and
flow velocity were imaged using a 7.5-MHz linear array transducer
ultrasound system (Apogee CX, Interspec ATL, Ambler, PA). Brachial
arterial diameter and blood flow velocity were recorded at an interval
of 1 min. After that, a blood pressure cuff was placed over the ipsilateral
upper arm just above the transducer and inflated for 5 min at 200 mm
Hg, then suddenly deflated. The postischemic scan was performed 60 s
after cuff deflation, and brachial artery diameter and flow were measured at 1-min intervals for 5 min. All images were recorded on videotape for subsequent off-line analysis on the same instrument by a
single observer blinded to the PON gene polymorphism.
Intraobserver variability for measuring brachial artery diameter and
flow was assessed by comparing a minimum of three separate baseline
measurements in each patient. The coefficient of variation for baseline
arterial diameter was 2.1%, and that for baseline arterial flow was 9.7%.
These values were not dissimilar from those reported by other researchers (17).
1079
the Hsp 92II in the presence of BSA (37 C over 3 h). The digested products
were separated by acrylamide gel (8%) electrophoresis and identified
ethidium bromide staining.
Analytical technique
All blood samples were drawn in dark test tubes to which lithium
heparin was added and immediately centrifuged at 4 C. The plasma
glucose concentration was determined immediately. All other blood
samples were frozen at ⫺20 C for further metabolite and hormone
determinations.
Plasma glucose was determined by the glucose oxidase method (glucose autoanalyzer, Beckman Coulter, Inc., Fullerton CA), and plasma
insulin was determined using a commercial double antibody solid phase
RIA [Linco Research, Inc., St. Charles, MO; coefficient of variation (CV),
4.8 ⫾ 0.2%; cross- reactivity with proinsulin, 0.2%]. Plasma HDL cholesterol was determined according to the method of Penttila et al. (20).
Commercial enzymatic methods were used in the determination of
serum total cholesterol (Monotest, Roche, Milan, Italy; CV, 3.6 ⫾ 0.7%)
(21) and triglycerides (Peridecrome, Roche; CV, 4.3 ⫾ 0.5%) concentrations (22). Serum low density lipoprotein (LDL) cholesterol levels were
calculated by the Friedwald formula (23). Plasma free fatty acid levels
were determined according to the method of Dole et al. (24).
Statistical analysis
All data were presented as the mean ⫾ sd. Data for the brachial artery
diameter and flow were expressed as percent changes from baseline
values calculated as the maximal change within the interval of observation. Changes in plasma triglycerides were calculated as thedifference
between Intralipid treatment and baseline values. The distribution of
paraoxonase genotypes frequencies was compared with the HardyWeinberg equilibrium model using the ␹2 test. The Pearson productmoment correlation was used. Paired t test allowed calculation of differences between baseline and Intralipid infusion within the same
genotype group. One-way ANOVA with Scheffè’s test was used to
analyze differences in clinical and laboratory findings among genotype
groups. Analysis of covariance allowed adjusting changes in diameter
and flow (either endothelium dependent or independent) for gender
ratio (categorized as M ⫽ 0 and F ⫽ 1), basal plasma triglycerides, and
changes in plasma triglycerides concentrations. P ⬍ 0.05 was chosen as
the level of significance.
Results
PON polymorphism screening
Baseline data
DNA was extracted from white cells according to the method of
Sambrook et al. (18) from cells obtained from a fasting blood sample. The
gene polymorphism (A-glutamine and B-arginine alleles) corresponding
to position 191 was analyzed by restriction isotyping using the procedure of Humbert et al. (19). Briefly, the primer was used to amplify this
polymorphic region. DNA (500 ng) was denatured at 94 C for 30 s,
annealing at 61 C for 30 s, and extension at 72 C for 30 s, with a final
extension time of 6 min. The PCR product (170 bp) was digested with
All subjects were adult and not obese. Analysis of the
distribution of the genotypes revealed that the frequency was
0.545 for the AA genotype, 0.356 for the AB genotype, and
0.099 for the BB genotype. When tested, such distribution
was compatible with the Hardy-Weinberg equilibrium. Anthropometric, clinical, and laboratory findings were similar
among genotype groups (Table 1). In particular, no signifi-
TABLE 1. Baseline anthropometric, clinical, and laboratory measurements among the different genotype groups
Age (yr)
Gender (M/F)
BMI (kg/m2)
FFM (kg)
FP glucose (mmol/L)
FP triglycerides (mmol/L)
FP FFA (␮mol/L)
FP total cholesterol (mmol/L)
FP LDL cholesterol (mmol/L)
FP HDL cholesterol (mmol/L)
AA (n ⫽ 55)
AB (n ⫽ 36)
BB (n ⫽ 10)
P
36.6 ⫾ 13.6
22/33
25.5 ⫾ 1.4
47.6 ⫾ 4.3
5.0 ⫾ 0.3
0.76 ⫾ 0.06
560 ⫾ 112
3.35 ⫾ 0.31
1.78 ⫾ 0.52
1.45 ⫾ 0.34
37.3 ⫾ 17.1
18/18
25.7 ⫾ 1.3
48.4 ⫾ 3.7
5.1 ⫾ 0.3
0.74 ⫾ 0.07
569 ⫾ 143
3.32 ⫾ 0.28
1.88 ⫾ 0.39
1.32 ⫾ 0.28
33.3 ⫾ 15.0
5/5
25.6 ⫾ 1.3
48.4 ⫾ 3.2
5.1 ⫾ 0.4
0.78 ⫾ 0.07
567 ⫾ 147
3.34 ⫾ 0.29
1.66 ⫾ 0.41
1.50 ⫾ 0.29
NS
NS
NS
NS
NS
NS
NS
NS
NS
All results are the mean ⫾ SD. All plasma metabolites were determined after an overnight fast. BMI, Body mass index; FFM, fat-free mass;
FP, fasting plasma; FFA, free fatty acids.
1080
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Vol. 86 • No. 3
PAOLISSO ET AL.
cant differences with regard to body mass index, fasting
plasma glucose, triglycerides, free fatty acids, or total, LDL,
and HDL cholesterol levels were found. In the whole population baseline brachial artery diameter and artery flow
were 3.09 ⫾ 0.34 mm and 73.9 ⫾ 17.9 mL/min, respectively,
without significant differences among the genotype groups.
Reactive hyperemia provoked by occlusion of the distal forearm resulted in a significant increase in brachial artery diameter (8.4 ⫾ 4.0%; P ⬍ 0.001) and brachial artery flow (8.4 ⫾
13.0%; P ⫽ 0.008) in all subjects studied without differences
among the three genotype groups (Table 2).
Intralipid study
In the Intralipid study similar and significant increases in
plasma triglycerides and free fatty acid concentrations in all
genotypes was found (Table 2). As far as brachial artery
diameter and flow are concerned, Intralipid infusion was
associated with a significant reduction in flow-induced
change in diameter in all genotype groups. Nevertheless,
subjects with the BB genotype had the strongest reduction in
this parameters compared with subjects with the AA and AB
genotypes (P for trend ⬍ 0.001; Table 2). In contrast, changes
in brachial artery flow from baseline values were similar in
all genotype groups (Table 2). At the end of Intralipid infusion and after adjusting for gender ratio, basal triglycerides,
and changes in plasma triglycerides concentrations, subjects
sharing the BB genotype still had the lowest changes in
diameter (0.8 ⫾ 0.4%) compared with AA (2.8 ⫾ 1.0%) and
AB (2.6 ⫾ 0.9%) genotypes (P for trend ⬍ 0.001). In contrast,
changes in flow among the three genotype were not significant despite adjustment for gender ratio, basal triglycerides,
and changes in plasma triglycerides concentrations (data not
shown).
At baseline, arterial blood pressure (115 ⫾ 3.5/80 ⫾ 1.8
mm Hg) was not different among the three groups. At the
end of the Intralipid study blood pressure did not significantly change compared with baseline values and did not
differ among the three study groups.
No significant correlation between flow-induced vasodilatation and baseline fasting plasma triglycerides, free fatty
acid, and total cholesterol levels in any genotype group was
found (data not shown).
NTG study
The 55 subjects studied were distributed among the 3
different PON genotypes; in particular, 25 subjects shared the
AA genotype, 20 shared the AB genotype, and 10 shared the
BB genotype. As shown in Table 3, NTG administration was
associated with a significant increase in brachial artery diameter in all genotype groups. Nevertheless, after Intralipid
infusion, a significant reduction in NTG-induced vasodilatation was found in all 3 groups, with the strongest reduction
in the subjects sharing the BB genotype (P for trend ⬍ 0.001
In contrast, NTG-induced changes in brachial artery flow
from baseline values were not significant after administration of NTG alone or after Intralipid infusion plus NTG). At
the end of Intralipid infusion and after adjusting for gender
ratio, basal triglycerides, and changes in plasma triglycerides
concentrations, subjects sharing the BB genotype still had the
lowest changes in diameter (7.4 ⫾ 0.9%) compared with AA
(13.1 ⫾ 3.2%) and AB (11.2 ⫾ 2.9%) genotypes (P for trend ⬍
0.001). In contrast, changes in flow among the three genotypes were still not significant despite adjustment for gender
ratio, basal triglycerides, and changes in plasma triglycerides
concentrations (data not shown).
Discussion
Our study demonstrates the occurrence of a more severe
endothelial dysfunction, as brachial reactivity, after acute
hypertriglyceridemia in healthy subjects with PON BB genotype than in those sharing the AA or AB genotype.
A major determinant of endothelium function and brachial
reactivity is the oxidative stress, which might contribute to
quench nitric oxide (14). Hypertriglyceridemia has been
shown to be a prooxidant factor (11) and to impair the endothelial response in healthy subjects (5). How plasma oxidative stress levels are regulated is still debated. More recently, it has been outlined that paraoxonase, the product of
the PON genes, inhibits LDL and HDL oxidation and preserves their function (4). To efficiently participate in the protection of LDL particles against oxidative modification, PON
should be present locally in the arterial wall, where the attack
by oxidizing radicals is currently thought to occur (3, 4). A
coordinate increase in paraoxonase as well as in apolipoprotein J/apolipoprotein A-1 complex in human aortic tissue
during the development of atherosclerosis has been also
documented by immunolocalization and has been interpreted as a sign of an adaptive process whose role is to
protect the artery wall from the increasing oxidative stress
associated with the progress of atherosclerosis (3). Furthermore, PON was found to use efficiently not only lipoproteinassociated peroxides but also hydrogen peroxides, which are
TABLE 2. Metabolic and hemodinamic variables at baseline and at the end of intralipid infusion in the population studied divided by
genotype groups
AA (n ⫽ 55)
Plasma triglycerides
(mmol/L)
Plasma FFA
(␮mol/L)
Change in diameter
(%)
Change in flow (%)
AB (n ⫽ 36)
BB (n ⫽ 10)
Baseline
P
Intralipid
Baseline
P
Intralipid
Baseline
P
Intralipid
0.76 ⫾ 0.06
0.001
2.73 ⫾ 0.43
0.74 ⫾ 0.07
0.001
2.82 ⫾ 0.56
0.78 ⫾ 0.07
0.001
2.83 ⫾ 0.46
560 ⫾ 112
0.001
1310 ⫾ 323
569 ⫾ 143
0.001
1219 ⫾ 335
567 ⫾ 146
0.001
1333 ⫾ 286
9.0 ⫾ 4.3
0.001
2.5 ⫾ 1.2
8.4 ⫾ 3.6
0.001
2.5 ⫾ 1.1
7.1 ⫾ 3.8
0.001
1.1 ⫾ 0.6a
8.9 ⫾ 14.0
NS
5.3 ⫾ 9.3
6.3 ⫾ 11.9
NS
5.2 ⫾ 6.5
13.1 ⫾ 10.7
NS
8.4 ⫾ 7.5
All results are the mean ⫾ SD. FFA, Free fatty acids.
Statistically significant difference at the end of intralipid infusion among different genotype groups was P ⬍ 0.001 for trend.
PON POLYMORPHISM AND BRACHIAL REACTIVITY
1081
TABLE 3. NTG-mediated changes in diameter and flow at baseline and after intralipid infusion in different PON genotypes
AA (n ⫽ 25)
Change in diameter
(%)
Change in flow (%)
AB (n ⫽ 20)
BB (n ⫽ 10)
NTG
P
NTG⫹intralipid
NTG
P
NTG⫹intralipid
NTG
P
NTG⫹intralipid
21.2 ⫾ 5.9
0.001
12.5 ⫾ 4.1
19.7 ⫾ 4.3
0.001
10.3 ⫾ 5.6
20.6 ⫾ 6.4
0.001
8.3 ⫾ 3.7a
12.5 ⫾ 9.4
NS
10.6 ⫾ 8.3
13.6 ⫾ 8.9
NS
11.1 ⫾ 7.4
11.3 ⫾ 7.1
NS
9.1 ⫾ 8.2
All results are the mean ⫾ SD.
a
Statistically significant difference at the end of intralipid infusion among different genotype groups was P ⬍ 0.001 for trend.
the major free radicals produced by the arterial wall cells
during atherogenesis and are responsible for LDL oxidation
(4). The ability of PON to hydrolyze (in addition to peroxides) may thus play an important role in eliminating potent
oxidants that are involved in atherosclerosis (4). Interestingly, the antioxidant effect of paraoxonase enzyme activity
is associated with different gene polymorphisms. In fact, the
PON BB genotype is associated with lesser protection of LDL
against the accumulation of lipid peroxides (25) and more
frequent CHD (3, 4). Our data provide strong physiopathological support for a role for the PON genotype in the development of CHD, as we demonstrated that adult healthy
subjects sharing the PON BB genotype have impaired vascular reactivity and thus are more prone to endothelium
dysfunction. Such impaired vascular reactivity, evident after
transient acute (5) and chronic (26) hypertriglyceridemia and
reversed by atorvastastin administration (26), is associated
with a significant increase in oxidative stress (11). Thus, we
can hypothesize that subjects with the PON BB genotype
have a lower antioxidant capacity and therefore are more
exposed to the risk of atherosclerosis as demonstrated by the
lower vascular reactivity in the presence of a prooxidant
factor. Such a hypothesis is also supported by results showing that subjects with the PON BB genotype have a greater
accumulation of lipid peroxides than those with the AA or
AB genotype when exposed to prooxidant factors (25, 27).
Noteworthy, in our data hypertriglyceridemia impaired
brachial artery vasodilatation in an endothelium-dependent
and -independent manner, and the subjects sharing the BB
PON polymorphism had worse vasodilatation in both of
these experimental conditions. Thus, one can hypothesize
that hypertriglyceridemia, via a free fatty acid-mediated increase in oxidative stress, might impair both the production
and the action of nitric oxide at endothelium level. This
hypothesis is also in agreement with previous data (5). In our
study we found that subjects sharing the BB genotype after
Intralipid infusion had a significant decrease from baseline
values in changes in brachial artery diameter, but not in flow.
Indeed, such an apparent discrepancy might be explained by
an increase in local blood pressure, which, in turn, had no
systemic effect, as demonstrated by the absence of difference
in change in systemic arterial blood pressure among the three
genotype study groups. Alternatively, one can hypothesize
that brachial arterial wall thickness was able to magnify the
difference in diameter and hide the changes in flow.
Despite the well known association among PON activity,
CHD, and oxidative stress, our study has some potential
limitations. Some subjects (n ⫽ 35) were light smokers, and
a smoking habit might affect our results. Indeed, looking at
the distributions of such subjects in the 3 genotype groups,
we found that only 1 subject had the BB genotype, whereas
20 and 12 had the AA and AB genotypes, respectively. Such
a distribution makes it unlikely that a smoking habit might
have influenced our data. The relationship between lower
vascular reactivity and more elevated oxidative stress in the
subjects with the BB genotype is apparently in contrast with
the evidence that the PON BB genotype is associated with the
strongest enzymatic activity against paraxon (28). Notwithstanding, the results reported by Mackness et al. (25) clearly
indicate that the activity of the enzyme against such a nonphysiological substrate as paraoxon cannot be interpreted as
directly predictive of a specific level of cardiovascular risk.
The use of an artificial lipid emulsion such as Intralipid for
transiently increasing plasma triglycerides and free fatty acid
concentrations is a further possible limitation of our study.
Another approach might be to examine the brachial reactivity after the ingestion of the fatty meal, as reported by Vogel
et al. (29). However, these researchers did not exclude the
effect of vasoactive hormones, such as insulin or intestinal
peptides, which increase after a fatty meal and are not affected by Intralipid infusion at least at the rate used in our
study. Furthermore, the rise in plasma triglycerides and free
fatty acid concentrations is much more reproducible after the
infusion of Intralipid than after a fatty meal, as also demonstrated by the fact the steady state plasma triglycerides
and free fatty acid levels were similar in all genotype groups.
In conclusion, our study shows that transient hypertriglyceridemia decreases vascular reactivity more in subjects
with the PON BB genotype than in those with the other
genotypes. Such data seem particularly important in light of
the fact that the PON BB genotype has been associated with
a more elevated frequency of CHD and that impaired vascular reactivity is an early sign of endothelium dysfunction,
which is normally associated with atherosclerosis. Further
studies will need to investigate whether peripheral arteries
and coronaries have a similar response to elevated plasma
triglycerides and free fatty acids and whether transient and
chronic hypertriglyceridemia have similar effects on vascular reactivity.
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IOF-Servier Young Investigator Fellowship
The International Osteoporosis Foundation is pleased to announce the joint winners of the first IOF-Servier
Young Investigator Fellowship: Dr. Freda Wynne of the University College of Cork and Dr. Luigi Gennari
of the University of Florence.
Call for applications: The next Fellowship will be awarded during the IOF-World Congress on Osteoporosis in Lisbon, May 2002. Financed by an unrestricted grant offered by Servier Research Group, the
IOF-Servier Young Investigator Research Fellowship is an award of 40,000 Euros to encourage young
scientists under the age of 40 to engage in high-quality research in the field of osteoporosis.
Deadline for submission of applications: January 15, 2002. For eligibility criteria and application forms
please see the IOF web site at www.osteofound.org or contact the IOF secretarial office (info@ioflyon.org)
at: International Osteoporosis Foundation, 71 cours Albert Thomas, 69447 Lyon Cedex 03 France. Phone: 33
4 72 91 41 77.