Atherosclerosis 214 (2011) 480485

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Atherosclerosis
journal homepage: www.elsevier.com/locate/atherosclerosis

Homocysteine is a determinant of ApoA-I and both are associated with ankle

brachial index, in an Ambulatory Elderly Population
Rosa Maria Guant-Rodriguez a,,1 , Rosario Spada b,1 , Maira Moreno-Garcia a,1 , Guido Anello b ,
Paolo Bosco b , Laurent Lagrost c , Antonino Romano b , Maurizio Elia b , Jean-Louis Guant a,b
a
Inserm U954, Medical Faculty and CHU of Nancy, University Henri Poincar, Nancy, France
b
IRCCS of Mental Retardation and Brain Aging, Oasi Maria S.S., Troina, Sicily, Italy
c
INSERM UMR866, Facult de Mdecine, Dijon, France

a r t i c l e i n f o a b s t r a c t

Article history: Objective: The ankle brachial index (ABI) is an indicator of lower extremity peripheral arterial disease
Received 29 September 2010 (PAD) and a predictor of atherothrombosis. ApoA-I and HDL are associated with PAD, in humans. Homo-
Received in revised form 7 November 2010 cysteine inuences the liver expression of ApoA-I and decreases its blood level and HDL in genetic
Accepted 19 November 2010
mice models. We aimed therefore to evaluate whether homocysteine and its nutritional determinants,
Available online 26 November 2010
folate and vitamin B12 are associated with ABI by inuencing HDL metabolism, in an ambulatory elderly
population.
Keywords:
Methods: 667 elderly volunteers from rural Sicily were assessed for ABI, homocysteine and its determi-
Ankle brachial index
Homocysteine
nants, lipid markers and other predictors of PAD. HDL size was assessed in 15 sera in upper and lower
HDL quartiles of Hcy distribution.
Folate Results: In multivariate analysis, ApoA-I and homocysteine were two predictors of ABI (-
Vitamin B12 coefcient = 2.86, P < 0.004 and -coefcient = 3.41, P < 0.001, respectively). Homocysteine correlated
negatively with ApoA-I (R = 0.147, P < 0.001) and with HDL-Cholesterol (R = 0.113, P = 0.003). The asso-
ciations of homocysteine, vitamin B12 and methylmalonic acid with ApoA-I and HDL2a particles and that
of homocysteine with increased small size HDL3c suggested mechanisms related with impaired synthesis
of ApoA-I and HDL and abnormal maturation of HDL particles.
Conclusion: The inuence of homocysteine on ApoA-I and HDL metabolism provides new insights on its
role on vascular diseases, at a cross-point between atherosclerosis and atherothrombosis.
2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction thrombosis and vascular disease [9]. Since then, epidemiological

Coronary artery and cerebrovascular diseases, together with
peripheral arterial disease (PAD), are responsible for the deaths
of more adults than any other medical condition in developed
countries [1] and are strongly related to age, gender and ethnicity,
dyslipidemia and inammation [28]. The ABI is a measure-
ment that provides objective data for the diagnosis of lower
extremity PAD [3]. Patients with a low ABI have a substantially
increased risk of death and severe vascular and thrombotic events
[3].
Homocysteine (Hcy) was rst implicated as a risk factor when
it was noticed that homocystinuric patients had a propensity to
Corresponding author at: University Henri Poincare, Inserm U954, Medical Fac-
ulty and CHU of Nancy, 54500 Vandoeuvre les Nancy, France.
Tel.: +33 383154484; fax: +33 383683279.
E-mail address: rm.rodriguez@chu-nancy.fr (R.M. Guant-Rodriguez).
1
These authors contributed equally to this work.
studies have shown an association of elevated plasma Hcy with
cerebro-vascular and coronary artery diseases and with left ven-
tricular dysfunction [1013]. A recent meta-analysis concluded that
patients with PAD have an elevated blood level of Hcy [14]. Hyper-
homocysteinemia increases cardiovascular morbidity by various
mechanisms that include oxidant and cellular stress, inammation,
endothelium dysfunction and thrombosis [10,11,15,16]. However,
the association of homocysteine with PAD and ABI needs to deserve
further attention, in particular concerning its potential inuence
on lipid metabolism. Two recent studies with mthfr-decient or
cbs//apoE/ mice showed that the genetically increased Hcy
alters the liver expression of ApoA-I and decreases the blood level of
ApoA-I and HDL-cholesterol (HDL-C) [17,18]. We aimed therefore
to evaluate whether homocysteine and its nutritional determi-
nants, folate and vitamin B12 exert deleterious effects on peripheral
arteries by inuencing ApoA-I and the HDL-dependent transport
of cholesterol. We investigated this hypothesis in an ambulatory
elderly population of 667 volunteers, which were systematically
assessed for ABI.

0021-9150/$ see front matter 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.atherosclerosis.2010.11.031
 R.M. Guant-Rodriguez et al. / Atherosclerosis 214 (2011) 480485
2. Methods
2.1. Setting and participants
San Teodoro and Cesaro are two rural villages of approximately
1500 inhabitants every one in the territory of Mounts Nebrodi,
Sicily (1150 m). In 2005, all the elderly between 60 and 85 years
old were invited to participate in the study. The number of the
enrollable subjects was 955, including 538 females and the nal
number of volunteers 667 (70.3%), including 400 females. The study
was conducted door-to-door and undertook clinical examination
and personal interview by physicians on clinical conditions, use of
medications, signs and symptoms of diseases and their risk factors.
Diagnosis of peripheral arterial disease (PAD) was made according
to established criteria [3,19]. All subjects provided written consent
and the local ethic committee (IRCCS Oasi Maria S.S., Troina, Sicily)
approved the study protocol.
2.2. Measurement of ABI
The ABI was performed as recommended [3], using a hand-
held Doppler probe (V2008, with a 8 MHz probe, GIMA, Gessate,
Italy). The systolic blood pressure was recorded from both brachial
arteries and from both the dorsalis pedis and posterior tibial arter-
ies after the patient has been at rest in the supine position for
10 min. Both arm pressures were recorded. If the arm blood pres-
sures were not equal, then the higher blood pressure was used
for subsequent blood pressure ratio calculations. We considered
ve ABI categories, ABI <0.90 (denite PAD), 0.900.99 (border-
line), 1.001.09 (low), 1.101.29 (normal), equal or superior to 1.30
(high), as described [19].
2.3. Blood collection and biological experiments
All participants underwent blood sample collection. Venous
blood from fasting subjects was immediately centrifuged and
stored at 20 C until analysis of vitamin B12 and folate.
Other parameters were immediately assayed. Hcy and methyl-
malonic acid (MMA) were determined by UPLCMS/MS, with
an Acquity UPLC BEH C18 column (1.7 m, 2.1 mm 50 mm,
Waters Corporation) [20]. Folate, vitamin B12, HDL-C, creati-
nine and other biological parameters were assayed as described
[12,2022]. The Friedewald formula was used to calculate
the low-density lipoproteincholesterol (LDLC) concentration
except in 2 subjects with serum triglyceride levels greater than
8.0 mmol/l [23]. Apolipoproteins A-I and B were measured by
immunonephelemetry (Siemens Co., Ltd., France). HDL size dis-
tribution (HDL2b, 9.7112.90 nm; HDL2a, 8.779.71 nm; HDL3a,
8.178.77 nm; HDL3b, 7.768.17 nm; HDL3c, 7.217.76 nm) was
assessed by polyacrylamide 1.525% gradient gel electrophoresis
(PAGE) in 15 serum samples, including 8 in the upper and 7 in the
lower quartile of Hcy, as previously described [24].
2.4. Statistics
Continuous data were summarized as mean SD or median
with 2575th percentiles. Prevalence was reported as percentage.
MannWhitneys U-test evaluated differences. Multivariate corre-
lation was tested by multiple regression analysis. For variables with
skewed distribution, log-transformation was done before multi-
ple regression analysis. Such transformation was applied to Hcy.
The associations between blood markers and ABI were evaluated
using analyses of covariance, adjusting either for sex or for sex,
smoking and diabetes mellitus. P-values <0.05 were considered sig-
nicant. Considering the limited size of the borderline ABI category
(n = 5), we evaluated these associations by grouping either category
481
2 with category 1 or distinguishing both categories. Corrections for
multiple comparisons were made using the Bonferroni test. Anal-
yses were performed using Stata Software version 11.0 SE (Stata
Corporation, TX, USA).
3. Results
3.1. Baseline characteristics
The clinical and biochemical characteristics of the population
are presented in Table 1. The average age of the 667 participants was
71.5 years, 400 (60.0%) were women and 28.9% had cardiovascular
diseases, including stroke, transitory ischemic attack, angor and/or
myocardial infarction. The average ABI was 1.09 0.01. The preva-
lence of low ABI was 5.8% (n = 39). There was 1.5-fold more diabetes
mellitus and 2-fold more males and smokers in subjects with low
ABI, compared with those with a normal ABI (Table 1). In contrast,
the percentage of diabetes mellitus in the high ABI group was
similar to that reported in the whole population (Table 1). Inter-
mittent claudidation was reported in 13 (33%) of the 39 subjects
with low ABI. Hcy was strongly associated with folate (R = 0.258,
P < 0.001) and vitamin B12 (R = 0.253, P < 0.001), but neither with
creatinine (P = 0.972) nor with C-reactive protein (P = 0.572).
3.2. Predictors of ABI
The categories of ABI were positively associated (P for trend)
with SBP, DBP, ApoA-I, folate and vitamin B12. Conversely, a nega-
tive trend of ABI groups was noted with age, Hcy, C-reactive protein
and creatinine. Similar results were obtained when either grouping
category 2 with category 1 (Table 1) or distinguishing both cate-
gories (Table 1bis online). The P for trend of the association of ABI
categories with either SBP or DBP was less signicant when the
high ABI was excluded from the analyses, while the same level
of signicance was reported for the other associations (Table 1).
In univariate regression analysis, the predictors of ABI were, by
order of signicance, age, SBP, DBP, Log-Hcy, ApoA-I, creatinine,
C-reactive protein, folate and B12 (Table 2). The predictors were
age (P < 0.001), SBP (P < 0.001), DBP (P < 0.035), Log-Hcy (P < 0.001),
ApoA-I (P < 0.001) and folate (P = 0.010) when excluding the high
ABI group from the analysis. In the multivariate analysis of a sex-
adjusted model that included creatinine and the other signicant
predictors of univariate analysis, the remaining independent pre-
dictors of ABI were age, SBP, ApoA-I and Log-Hcy in the whole
population (Table 2). We also performed the multivariate analy-
sis in two models where ApoA-I and Log-Hcy were alternatively
excluded (Table 2). Log-Hcy was associated with ABI with P < 0.001
in the second model (without ApoA-I), and ApoA-I was associ-
ated with ABI with P < 0.001 in the third model (without Log-Hcy,
Table 2). The same conclusions were reached, when performing the
analyses after adjustment for sex, smoking and diabetes mellitus
(Table 2bis online). When excluding the high ABI group from the
analyses, the predictors were age (P < 0.001), Log-Hcy ( = 3.89,
P < 0.001, second model without ApoA-1) and ApoA-I ( = 3.67,
P < 0.001, third model without Log-Hcy). The same conclusion was
reached when excluding the cases with cardiovascular diseases
from the analysis.
3.3. Association of ApoA-I, HDL-C and HDL particle size with
homocysteine, methylmalonic acid and vitamin B12
Signicant negative correlations were observed between ApoA-
I and Hcy (P < 0.001) and between HDL-C and Hcy (P = 0.003) (Fig. 1).
The same signicance was obtained when excluding either the
high ABI group or the subjects with cardiovascular diseases from
the analyses. In univariate regression analysis, the determinants
482 R.M. Guant-Rodriguez et al. / Atherosclerosis 214 (2011) 480485

Table 1
Clinical and biological characteristics of the study population, according to the 5 groupsa of ankle brachial index (ABI).

Characteristic (%, All groups (n = 667) 12 (denitive and 3 (low/normal) 4 (normal) 5 (High) (n = 155) P-for trend all
median and borderline) (n = 44) (n = 164) (24.6%) (n = 304) (45.6%) (23.2%) groups/groups 14
interquartiles or (6.6%)
mean SD)

Men, n (%) 267 (40.0) 32 (72.7) 69 (42.1) 107 (35.2) 59 (38.1) <0.001/<0.001
Age (years) 71.5 0.3 76.1 1.0a 72.0 0.5 71.1 6.9 70.6 6.8 <0.001/<0.001
Systolic blood pressure 141 1 147 23a 147 21 142 20 130 17 <0.001/0.021
(mmHg)
Diastolic blood 78 1 78 14a 82 11 80 12 74 12 <0.001/0.947
pressure (mmHg)
Current smoker, n (%) 78 (11.7) 9 (23.1) 23 (14.0) 36 (11.8) 10 (6.5) 0.035/0.235
Diabetes mellitus, n (%) 153 (22.9) 17 (38.6) 35 (21.3) 64 (21.1) 37 (23.9) 0.025/0.011
Total cholesterol 202 (176226) 198 (164227) 204 (177226) 199 (172224) 206 (182235) 0.220/0.654
(mmol/L)
Triglycerides (mmol/L) 108 (79146) 115 (84182) 98 (74133) 110 (79150) 110 (831141) 0.953/0.800
HDL cholesterol 58 (4868) 59 (496) 58 (4670) 57 (4869) 58 (4969) 0.325/0.771
(mmol/L)
LDL cholesterol 118 (95141) 116 (83-138) 120 (97141) 114 (94137) 122 (85147) 0.373/0.954
(mmol/L)
ApoA-I (g/L) 1.7 (1.51.9) 1.5 (1.3-1.7) 1.6 (1.41.9) 1.7 (1.51.9) 1.7 (1.51.9) <0.001/<0.001
ApoB (g/L) 1.0 (0.91.2) 1.0 (0.91.1) 1.0 (0.91.2) 1.0 (0.91.2) 1.0 (0.91.2) 0.449/0.414
Homocysteine 14.5 (11.918.1) 19.8 (14.622.5) 15.3 (12.619.7) 14.0 (11.716.9) 13.5 (10.916.9) <0.001/<0.001
(mol/L)
Folate (nmol/L) 11.8 (8.715.8) 9.9 (7.213.5) 10.9 (8.316.1) 12.1 (9.216.0) 12.4 (9.316.3) 0.002/0.009
Vitamin B12 (pmol/L) 297 (209401) 237 (178391) 299 (213404) 283 (204404) 323 (240401) 0.018/0.222
Methylmalonic acid 0.15 (0.110.24) 0.23 (0.130.36) 0.16 (0.110.24) 0.15 (0.110.23) 0.14 (0.090.21) 0.014/0.018
(mmol/L)
Creatinine (mmol/L) 9 (8-11) 9 (811) 9 (811) 9 (811) 9 (811) 0.192/0.846
Albumin (g/L) 43 (4145) 42 (4144) 43 (4145) 43 (4145) 42 (4144) 0.425/0.632
C-reactive protein 2.8 (1.65.4) 2.3 (1.05.1) 3.3 (1.96.6) 2.6 (1.55.1) 2.9 (1.75.4) 0.615/0.727
(mg/L)
a
Groups of ABI: (1) <0.90 (denite PAD); (2) 0.900.99 (borderline); (3) 1.001.09 (low); (4) 1.101.29 (normal); (5) equal or superior.

of ApoA-I were albumin, Log -C-reactive protein, total cholesterol,
triglycerides, HDL-cholesterol, Log-MMA and Log-Hcy and in mul-
tivariate analysis, cholesterol, triglycerides, HDL-cholesterol and
Log-Hcy (Table 3). The determinants of HDL-C were serum cre-
atinine, albumin, total cholesterol, triglycerides, ALAT, Log-Hcy,
vitamin B12, ApoA-I, Log C-reactive protein and folate (P < 0.001,
P < 0.001, P < 0.001, P < 0.001, P = 0.003, P = 0.004, P = 0.009, P = 0.010,
P = 0.020 and P = 0.040, respectively). In multivariate analysis, the
independent predictors of HDL-C were cholesterol, triglycerides,
Log-Hcy and albumin (-coefcient = 11.8 and P < 0.001, = 13.9
and P < 0.001, = 2.3 and P < 0.001 and = 2.1 and P = 0.022,
respectively). We investigated further the inuence of Hcy on HDL
size distribution by PAGE in 15 subjects with contrasted concentra-
tion of Hcy. We observed an increased concentration of the small
size HDL3c in subjects with a concentration of Hcy exceeding the
upper-quartile, compared to those in the lowest quartile (Fig. 2). In
addition, a signicant correlation was evidenced between HDL2a
and vitamin B12.
4. Discussion
In this study of an ambulatory elderly population, the prevalence
of low ABI was similar to that observed in other community-based
studies [2,4,19]. We reported also a high incidence of cases with
high ABI, indicative of non-compressible arteries in elderly patients
[3]. These individuals may have arterial disease, despite high ABI.

Table 2
Sex adjusted univariate and multivariate analyses of the determinants of the ankle brachial index (ABI) in 667 elderly volunteers.

Determinant Univariate Multivariate

Correlation P-Value First model (with ApoA1 Second model (without Third model (without
and Log-homocysteine) ApoA1) Log-homocysteine)

Beta coefcient P-Value Beta coefcient P-Value Beta coefcient P-Value

Age 0.163 <0.001 2.74 0.006 2.63 0.009 3.22 0.001

Serum creatinine 0.102 0.008
Albumin 0.028 0.461
ApoA-I +0.136 <0.001 2.79 0.005 2.86 0.004
C-reactive protein 0.007 0.851
Total cholesterol +0.061 0.116
Triglycerides +0.005 0.895
HDL-cholesterol +0.066 0.089
LDL-cholesterol +0.034 0.378
ALAT 0.059 0.130
ASAT 0.051 0.191
Log-homocysteine 0.218 <0.001 2.41 0.016 3.41 <0.001
Folate +0.116 0.003
Vitamin B12 +0.114 0.003
Methylmalonic acid 0.109 0.060
Systolic blood pressure 0.324 <0.001 7.91 <0.001 7.67 <0.001 8.06 <0.001
Diastolic blood pressure 0.229 <0.001
 R.M. Guant-Rodriguez et al. / Atherosclerosis 214 (2011) 480485 483

Table 3
Sex adjusted univariate and multivariate analyses of the determinants of the plasma
level of ApoA-I in 667 elderly volunteers.

Determinant Univariate Multivariate

Correlation P-Value Beta coefcient P-Value

Age 0.031 0.455

Serum creatinine 0.085 0.038
Albumin +0.134 0.001
Log-C-reactive protein 0.143 0.001
Total cholesterol +0.377 0.001 3.43 0.006
Triglycerides 0.139 0.001 4.65 <0.001
HDL-cholesterol +0.762 <0.001 25.41 <0.001
LDL-cholesterol 0.012 0.772
ALAT 0.034 0.407
ASAT +0.059 0.152
Log-homocysteine 0.147 <0.001 3.08 0.002
Folates +0.034 0.411
Vitamin B12 +0.088 0.031
Methylmalonic acid 0.162 0.009

To undertake this diagnosis limitation, we evaluated also the asso-

Fig. 1. Correlation (Pearson analysis) between logarithmically transformed total
homocysteine (Log-Hcy) and either Apoprotein A1 (ApoA-I) (top) or HDL-
Cholesterol (HDL-C) (bottom). Dotted lines: 95% condence intervals of the slope.
ciation of ABI with determinants of Hcy and lipid metabolism after
exclusion of the subjects with high ABI and found results similar
to those obtained in the whole population.
A recent consensus review considered that Hcy could be a
stronger risk factor for PAD than for coronary artery disease [25].
Most of the risk factors of low ABI may inuence the Hcy blood level
[28,10], making difcult to establish whether it is a cause or a con-
sequence of low ABI or both. From this point of view, it should be

Fig. 2. (A) Box plots (median and quartiles) of the distribution (in percentage) of HDL particles in 15 serum samples, including 8 in the upper and 7 in the lower quartile of Hcy.
The size of HDL particles (HDL2a, 8.779.71 nm; HDL2b, 9.7112.90 nm; HDL3a, 8.178.77 nm; HDL3b, 7.768.17 nm; HDL3c, 7.217.76 nm) was assessed by polyacrylamide
1.525% gradient gel electrophoresis. (B) Correlation (Pearson analysis) between HDL2a and serum vitamin B12. Dotted lines: 95% condence intervals of the slop.
484 R.M. Guant-Rodriguez et al. / Atherosclerosis 214 (2011) 480485
noticed that Hcy was neither associated with creatinine nor with
C-reactive protein in our population. In agreement with a previous
study, we found an association of ABI with age, SBP, C-reactive pro-
tein, HDL and ApoA-I in univariate analysis [26]. The ABI was also
associated with the nutritional determinants of Hcy, folate and vita-
min B12 and with MMA, a metabolic marker of vitamin B12 cellular
status. Hcy and ApoA-I were the two independent predictors of ABI,
in the rst model of our multivariate analysis, showing that other
effects of Hcy than those inuencing apoA-I plasma levels may also
play a role in peripheral artery disease. The association of Hcy with
PAD has been clearly evidenced in a recent meta-analysis based on
14 studies [13].
In agreement with the association between ApoA-I and Hcy in
our elderly ambulatory population, a reduced expression of ApoA-I
was reported in mice with genetically induced hyperhomocys-
teinemia and in patients with coronary artery disease [17,18]. In
the mthfr+/ decient mice model, the reduced liver expression of
apoA-I resulted from the inuence of Hcy on the expression of per-
oxisome proliferator-activated receptor alpha (PPAR) and on the
related activity of ApoA-I promoter [18]. The association of vita-
min B12 and MMA with ApoA-I and HDL2a particles observed in
our study may reect an inuence of cellular B12 (needed for both
metabolisms of Hcy and MMA) on the synthesis and metabolism of
HDL particles. These associations should deserve further attention
in the light of recent data on the effects of the decreased cellular
availability of B12 on TNF and MAPK pathways and the inuence of
these pathways on ApoA-1 [20,27]. In addition to its inuence on
ApoA-I, we also observed that hyperhomocysteinemia was asso-
ciated with an increased concentration of the small size HDL3c.
This suggested an impaired HDL maturation via an ApoA-I related
mechanism, which was in agreement with the absence of large HDL
particles observed in hyperhomocysteinemic cbs/apoe/ mice
[17].
Most of the interventional studies that aimed to decrease the
risk of coronary artery diseases by lowering Hcy with vitamin B sup-
plements failed to nd a benet of the supplementation [28,29]. In
contrast, a recent randomized clinical trial that included folate sup-
plementation increased ABI and decreased the pulse wave velocity,
suggesting an inuence on lower extremity PAD [30]. These con-
trasting results suggest that Hcy exerts more critical effects in
arterial diseases of extremities than in those of large vessels, despite
the lack of knowledge about the mechanisms that underlie the
association between Hcy and PAD. Our data suggest that the inu-
ence of Hcy on ApoA-I and HDL metabolism could be one of
them.
5. Conclusions
Little is known about the mechanisms that underlie the associa-
tion of homocysteine with peripheral arterial disease. We showed
that Log-homocysteine and ApoA-I are two interacting biological
predictors of ankle brachial index in an ambulatory elderly popu-
lation. Taken together, the associations of homocysteine, vitamin
B12 and methylmalonic acid with ApoA-I and HDL2a particles and
that of homocysteine with small size HDL3c suggest mechanisms
related with the impaired synthesis of ApoA-1 and HDL and abnor-
mal maturation of HDL particles.
Acknowledgement
This study was nanced by the Region Lorraine and by a grant
from the national agency for research (ANR Nutrivigne). The
authors have no conicts of interests and disclosures of nancial
support to declare.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.atherosclerosis.2010.11.031.
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