Journal of Human Hypertension (2005) 19, 233–240

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ORIGINAL ARTICLE
Endothelial nitric oxide synthase
polymorphism, nitric oxide production,
salt sensitivity and cardiovascular risk
factors in Hispanics
IS Hoffmann1, R Tavares-Mordwinkin2, AM Castejon2, AB Alfieri1 and LX Cubeddu1,2
1
Center for the Detection and Treatment of Silent Risk Factors for Cardiovascular and Metabolic Disease,
Division of Clinical Pharmacology Unit, School of Pharmacy, Central University of Venezuela; 2Department
of Pharmaceutical Sciences, College of Pharmacy, Health Professions Division, NOVA Southeastern
University (NSU), Fort Lauderdale, FL, USA

Mutations in the endothelial nitric oxide synthase
(eNOS) gene may be associated with abnormal nitric
oxide (NO) production and cardiovascular diseases. In
this study, we investigated the prevalence of two eNOS
polymorphisms, the Glu298Asp variant on exon 7, and
the 4a/b variable number of tandem repeats (VNTR) on
intron 4, and their association with blood pressure (BP),
NO production, salt sensitivity and cardiovascular risk
factors in healthy Venezuelans. The prevalence of both
polymorphisms in Venezuelans was comparable to that
described for Caucasians, but significantly different
from that known for African-Americans and Japanese.
The 4a/b genotype was associated with reduced levels
of NO metabolites (25% decrease), larger BP lowering in
response to salt restriction (9.0 vs 4.8 mmHg, Po0.05),
greater prevalence of salt sensitivity (39% in 4a/b and
27% in 4b/b; Po0.05) and with higher LDL-cholesterol
levels. The Glu298T polymorphism did not affect NO
production, nor it was associated with salt sensitivity.
Glu298Asp polymorphism was positively associated
with higher weight, triglycerides and LDL-cholesterol.
Neither polymorphism was associated with changes in
fasting or postload serum glucose, BP, obesity and
albuminuria. In conclusion, the prevalence of eNOS
polymorphisms is strongly determined by ethnic fac-
tors. The 4a/b gene polymorphism could be a genetic
susceptibility factor for the BP response to salt intake
and for the genetic control of NO production. The
reduced NO production in subjects with the 4a/b
genotype may be responsible for the increased sensi-
tivity of their BP to salt.
Journal of Human Hypertension (2005) 19, 233–240.
doi:10.1038/sj.jhh.1001801
Published online 25 November 2004

Keywords: eNOS polymorphism; nitric oxide; salt sensitivity; blood pressure; dysmetabolic cardiovascular syndrome

Introduction encoding for the NO synthesizing enzymes might

Salt sensitivity is characterized by an exaggerated
blood pressure (BP) response to changes in salt
intake.1 Nitric oxide (NO) has been shown to play a
role in intrarenal haemodynamics, sodium home-
ostasis and salt-sensitive hypertension. In animal
models and in human subjects, salt sensitivity has
been shown to be linked to impaired NO produc-
tion.2–6 Therefore, and because of the well-known
role of NO on BP control, salt-sensitive hypertension
and vascular function, mutations in the genes
Correspondence: Dr LX Cubeddu, HPD, Department of Pharma-
ceutical Sciences, College of Pharmacy, NOVA Southeastern
University, 3200 S University Dr., Ft Lauderdale, FL 33328, USA.
E-mail: lcubeddu@nova.edu
Received 2 September 2004; revised 5 October 2004; accepted
6 October 2004; published online 25 November 2004
be associated with salt sensitivity and cardiovascu-
lar diseases.
Endothelial NO synthase (eNOS) is one of the
three isoforms of the NOS that can generate NO from
its precursor L-arginine. Despite much evidence
supporting the role for NO in salt sensitivity, prior
to this work no studies had been conducted on the
relationships between eNOS polymorphisms and
salt sensitivity. In the present study, we evaluated
the relationships between salt sensitivity and two
eNOS gene polymorphisms; namely: (a) the 894 G/T
substitutions in exon 7 of the eNOS gene, which
results in a glutamate or aspartate, respectively, at
codon 298 in the eNOS protein (Glu298Asp), and (b)
the 27 bp variable number of tandem repeats (VNTR)
polymorphism within intron 4 of the eNOS gene,
with the variant a allele with four tandem repeats
 eNOS polymorphisms and salt sensitivity
IS Hoffmann et al
234
and the b allele with five tandem repeats. In
addition, the association between eNOS polymorph-
isms and NO production, BP and cardiovascular risk
factors was also evaluated. Finally, the unknown
prevalence of eNOS polymorphisms in Venezuelans,
a previously nonstudied ethnic group, was investi-
gated.
Materials and methods
Study population
A total of 126 consecutive, otherwise healthy
subjects, attending our Center for the Detection
and Treatment of Silent Cardiovascular Risk Factors,
were evaluated for eNOS polymorphism, salt sensi-
tivity and cardiovascular risk factors. The exclusion
criteria were: age greater than 70, a history of angina
pectoris, myocardial infarction, congestive heart
failure, valvular heart disease, cerebral infarction
or haemorrhage, transient ischaemic attacks, arterio-
sclerosis obliterans, pulmonary disease; any patient
with active disease, evidences of renal or hepatic
dysfunction, urinary tract infection, active inflam-
matory disease states, severe hypertension, diabetes
mellitus; treatment with organic nitrates, women on
birth control pills and serum creatinine concentra-
tion greater than 2 mg/dl. Any medication was
discontinued at least 4 weeks before the sodium-
sensitivity protocol was started. The study was
approved by the Institutional review Boards of
NOVA Southeastern University and the University
Hospital of the Central University of Venezuela. All
participating subjects gave their informed consent.
Determination of salt sensitivity
Salt sensitivity testing was determined as previously
described.7 Briefly, subjects were placed on a high
salt intake for 7 days, followed by a low-salt intake
for an additional 7 days. On days 6 or 7 of both
sodium diets, patients returned to the Center for the
following procedures: systolic blood pressure (SBP),
diastolic blood pressure (DBP), 24 h sodium and
potassium excretion, 24 h nitrites and nitrates
excretion, serum and urinary creatinine levels. The
average of three BP determinations in supine
position was used for determining salt sensitivity.
Patients were classified as salt-sensitive, salt inter-
mediate and salt resistant. If the difference in mean
BP between high- and low-sodium weeks was equal
to or more than 10 mmHg, the patient was deemed
salt sensitive. Salt resistance was defined as in-
creases of less than 3 mmHg, no change or decreases
in mean BP. Subjects with BP increases greater than
3 but less than 10 mmHg were classified as of
intermediate sensitivity to salt.
Journal of Human Hypertension
Genetic analysis—eNOS Glu298Asp polymorphism
DNA was extracted from blood anticoagulated with
EDTA according to standard protocols. The G to T
substitution polymorphism occurring at nucleotide
position 894 of exon 7 in the eNOS gene, which
results in the genetic variant of amino-acid residue
298, was evaluated. Genotyping was performed with
polymerase chain reaction (PCR). Two allele-speci-
fic primers, FP 50 -TCC CTG AGG AGG GCA TGA
GGC T-30 and RP 50 -TGA GGG TCA CAC AGG TTC
CT-30 , were used in a single-tube reaction assay. The
PCR was performed on a Perkin–Elmer Thermo-
cycler in a 50 ml reaction mixture, containing 5 ml of
genomic DNA solution, which contained 0.5 mmol/l
of each primer, MgCl2-free Thermophillic DNA
Polymerase Reaction buffer, 200 mmol/l of PCR
Nucleotide Mix, 1.25 U Ampli-Taq DNA polymerase
(Promega, Madison, WI, USA) and 1.5 mmol/l
MgCl2. The conditions were initial denaturation for
5 min at 941C followed by 45 cycles of denaturation
for 1 min at 941C, annealing for 1 min at 611C and
extension for 1 min at 721C. The size of the PCR
product was 457 bp and was used in the restriction
enzyme assay. BanII (restriction enzyme), 10 U, were
added to the PCR product and incubated at 371C for
at least 20 h. The BanII digested the alleles contain-
ing G at nucleotide position 894 into two fragments
of 137 and 320 bp, while leaving the DNA intact
when T was present. The DNA was then resolved on
a 2% agarose gel (Promega), stained with ethidium
bromide and visualized under UV light. Subjects
homozygous for the 298Glu or 298Asp alleles were
identified as G/G or T/T, respectively. Subjects
heterozygous for the polymorphism were identified
as G/T.
Genetic Analysis—eNOS 4a/b polymorphism
DNA was extracted from blood anticoagulated with
EDTA according to standard protocols. The 27 bp
VNTR polymorphism within intron 4 of the eNOS
gene, with the variant a allele with four tandem
repeats and the b allele with five tandem repeats,
were quantified. Genotyping was performed with
PCR. Two allele-specific primers, FP 50 -AGG CCC
TAT GGT AGT GCC TTT-30 and RP 50 -TCT CTT AGT
GCT GTG GTC AC-30 , were used in a single-tube
reaction assay. The PCR was performed in a 30 ml
reaction mixture, containing 5 ml of genomic DNA
solution, which contained 0.25 mmol/l of each
primer, MgCl2-free Thermophillic DNA Polymerase
Reaction buffer, 200 mmol/l of PCR Nucleotide Mix,
1.25 U Ampli-Taq DNA polymerase and 1.5 mmol/l
MgCl2. The conditions were initial denaturation at
951C for 5 min, followed by 35 cycles of denatura-
tion for 50 s at 951C, annealing for 1 min at 471C and
extension for 1 min at 721C. The size of the PCR
products were 393 bp for the DNA containing the
a allele and 420 bp for the DNA containing the
 eNOS polymorphisms and salt sensitivity
IS Hoffmann et al
235
b allele, which were resolved on a 3% agarose gel. No differences in the BP response to changes in
After staining with ethidium bromide, PCR products salt intake were observed in G/G and G/T subjects
were visualized under UV light. Subjects homozy- (Figure 1, Table 2). In addition, a similar percentage
gous for the a and b alleles were identified as 4a/a of salt sensitivity was found in the G/G and G/T
and 4b/b, respectively, while heterozygous subjects groups (Table 2). Changes in salt intake during the
were identified as 4a/b. salt sensitivity testing were reflected by changes in
urinary excretion of sodium (Table 3). No significant
differences in sodium excretion were observed
Determination of nitrites and nitrates in urine samples between the groups of subjects when placed on
high- or low-sodium diets. Similarly, no differences
Nitrites plus nitrates were quantified employing a
modification of a commercially available NO assay
kit (Oxford Biomedical research Inc., Oxford, MI, Table 1 Glu298Asp polymorphism: prevalence and patient
characteristics
USA).5 Urine samples (24 h) were collected at the
end of the high- and low-sodium diets (10 days G/G G/T+T/T
restriction from nitrate and nitrite containing foods).
Nitrates present in the urine supernatant were Subjects n (%) 55 (51.4) 52 (48.6)
reduced 1:1 to nitrites by incubation with metallic M/F 39/16 27/25
cadmium beads for 24 h. The total nitrite concentra- Age (years) 4071.6 4171.6
Weight (kg) 7672.1 8072.0
tion was then estimated by the Griess reaction, using WHR 0.8870.01 0.970.01
a multiwell microplate for reading of sample BMI (kg/m2) 2970.7 29.570.6
absorbance at 540 nm. This value represents the SBP (mmHg) 11872 12272*
total amount of urine NO end products (nitrite þ DBP (mmHg) 7972 8071.4
FSG (mg/dl) 9572 9873
nitrate). Urine samples were processed in dupli- AUC glucose (mg/dl h2) 68711 85710
cates. Total cholesterol 20776 222.177
LDL-C 13676 14978.3*
HDL-C 4372 4272.2
TG 135710 160717*
Statistics and data analysis UAE 10.172 9.371
Categorical variables were compared by means of
G/G: Glu298Glu; G/T: Glu298Asp; T/T: Asp298Asp after a 75 g oral
the w2 test. Continuous variables were compared by load of glucose in mg/dl h2. Total cholesterol, LDL-C, HDL-C and
the Student’s t-test for independent samples and triglycerides in mg/dl. UAE: urinary albumin excretion in mg/24 h.
paired t-test for paired samples or by a one-way *Po0.05.
ANOVA followed by a Duncan’s test. Genetic
equilibrium was confirmed by the Hardy–Weinberg G/T a/b
criterion and was analysed by the w2 test. Results are + +
shown as mean values7s.e.m.; differences were G/G T/T b/b a/a
0
considered significant at values of Po0.05. The
assumption used for power calculation required a
sample size of 50 subjects per group, which we have
used (wild vs mutant) to provide an 80% power to
detect a 30% reduction in the urinary excretion of
(SBP low salt - SBP on high salt)
Change in SBP (mmHg)

NO metabolites with a 5% type I error rate for a one- -4

sided test.

Results
eNOS Glu 298Asp polymorphism
We found that 42% of subjects screened were G/T,
6.6% were homozygous to the mutation (T/T), and
51.4% carried the wild-type genotype (G/G)
(Table 1). The sample examined was in the Hardy–
Weinberg equilibrium. Subjects with the T allele (G/
T þ T/T) had higher levels of LDL-cholesterol
(P ¼ 0.01) and of triglycerides, and higher SBP than
those carrying the wild-type gene (P ¼ 0.03) (Table 1).
No significant differences were observed for the age,
SBP, DBP, fasting serum glucose, HDL-cholesterol
and urinary albumin excretion levels between both
groups (Table 1).
-8
*
-12
Figure 1 Effects of Glu298Asp and 4 a/b eNOS polymorphisms on
salt sensitivity. Comparative changes in SBP in individuals
carrying the wild (G/G) and the mutated (G/T þ T/T) genotype,
and for the 4b/b and 4a/b genotypes during the salt-sensitivity
testing and mean values7S.E.M are shown. Data are expressed as
the reduction in SBP experienced when going from a high- to a
low-salt intake. **Significantly different from 4b/b at Po0.01.

Journal of Human Hypertension

 eNOS polymorphisms and salt sensitivity
IS Hoffmann et al
236
Table 2 Glu298Asp polymorphism and salt sensitivity b/b
1250
a/b
G/G n (%) G/T+T/T n (%) Total n

SS 17 (31%) 18 (34.6) 35 1000

Urinary NO metabolites
SI 13 (23.6) 11 (21.1) 24
SR 25 (45.4) 23 (44.2) 48 *
**

(mmoles/day)
Total n 55 52 107 750 **

SS: salt sensitive; SI: intermediate sensitivity to salt; SR: salt

resistance.
500

Table 3 Glu298Asp polymorphism: BP and electrolyte excretion

during high and low salt intake 250

G/G G/T+T/T
0
Subjects (n) 55 52 39 ± 4 130 ± 3 310 ± 6
HNa+-SBP (mmHg) 12272 12472 (low salt intake) (customary salt (high salt intake)
HNa+-DBP 8071.6 8171.5 intake)
UNa+/day 308711 320720
LNa+-SBP 11571.7 11972 Urinary sodium excretion
LNa+-DBP 7771.3 7971.4 (mEq /day)
UNa+/day 3773 3775
Figure 2 eNOS 4a/b polymorphism and NO metabolite excretion.
D SBP (mmHg) 6.071.2 6.571.6
Change in the urinary excretion of NO metabolites at three levels
D DBP (mmHg) 3.070.8 3.071.2
of salt intake are shown. Results are expressed as NO metabolite
D-UNa+/day 269715.3 285726
in mmol/day. Abscissa: three levels of sodium excretion.
Significant differences between 4b/b and 4a/b at *Po0.05 and
UNa+: urinary sodium excretion in mEq/day during a high-sodium **Po0.01.
diet (HNa+) and a low-sodium diet (LNa+).
Systolic and diastolic BP in mmHg.
HNa+- SBP and DBP: BP during a high-sodium diet.
Table 4 4a/b polymorphism: prevalence and patient character-
LNa+- SBP and DBP: BP during a low-sodium diet.
D SBP was calculated as SBP during high salt minus SBP during low
istics
salt.
D DBP was calculated as DBP during high salt minus DBP during low b/b a/b+a/a
salt.
D-UNa+/day was calculated as UNa+/day during high salt minus Subjects (n) 82 44
UNa+/day during low salt. M/F 35/47 17/27
Age (years) 3971.6 4171.5
Weight (kg) 79.572 75.572.1
in the urinary excretion of potassium were observed WHR 0.8970.01 0.8970.01
between groups (data not shown). BMI (kg/m2) 29.470.6 28.670.8
SBP (mmHg) 11971.7 12172
The urinary excretion of NO metabolites was DMP (mmHg) 7971 80.371.9
quantified during customary, high and low levels FSG (mg/dl) 9671.8 9473.8
of salt intake. There were no significant differences AUC (glucose mg/dl h2) 8079 65713.5
in the urinary excretion of NO metabolites between Total cholesterol 20876 22078.6
the G/G and the G/T groups (Figure 2). At customary LDL-C 13376 15177.2*
HDL-C 4272 4171.9
sodium intake (B130 mEq/day), the urinary excre- TG 156714 142713
tion of NO metabolites averaged 9787103 mmol/day UAE 1072 1172
in the G/G group and 9387108 mmol/day in the
subjects with the T allele (G/T þ T/T). Under condi- eNOS (a/b): 27 bp repeat in intron 4 (b: 5 repeats; a: 4 repeats).
tions of high salt intake and of salt restriction, the FSG: fasting serum glucose in mg/dl. AUC-glucose: area under the
curve for glucose after a 75 g oral load of glucose in mg/dl h2. Total
urinary excretion of NO metabolites averaged cholesterol, LDL-C, HDL-C and triglycerides in mg/dl. UAE: urinary
781797 and 817780 mmol/day, respectively, in albumin excretion in mg/24 h.
the G/G group, and 8817110 and 8807109 mmol/ *Po0.05.
day, respectively, in the G/T þ T/T group (P40.1).
equilibrium. Higher levels of LDL-cholesterol were
found in the 4a/b group than in the 4b/b group of
eNOS intron 4 polymorphism individuals (P ¼ 0.01) (Table 4). No significant
differences were observed in the age, SBP, DBP,
The frequency of the 4a/b VNTR polymorphism (4 or fasting serum glucose, glucose levels during an oral
5 repeats of 27 bp, respectively) in intron 4 of the tolerance test, HDL-cholesterol, triglycerides and
eNOS gene was evaluated (Table 4). We found that urinary albumin excretion levels of both groups.
65.1% of the subjects screened were 4b/b, 34.1% The BP response during the salt sensitivity testing
were 4a/b and only one subject (0.8%) was found was quantified. Shifting from a high- to a low-salt
4a/a. The sample examined was in Hardy–Weinberg diet produced greater reductions in SBP in subjects

Journal of Human Hypertension


carrying the 4a/b genotype than in those carrying the
4b/b genotype (Figure 1, Table 5). In addition, more
subjects were found to be salt sensitive in the 4a/b
(39%) than in the 4b/b group (27%) (Po0.05). There
were no significant differences in the amounts of
sodium excreted by 4b/b and 4a/b subjects when
either on high- or low-sodium diets (Table 6).
Similarly, no differences in the urinary excretion
of potassium were observed between both groups
(data not shown).
The daily urinary excretion of NO metabolites,
expressed either as mmol/24 h or corrected for
urinary creatinine (mmol/mg creatinine) (not shown),
was significantly lower in the 4a/b group than in
the 4b/b group (Po0.01) (Figure 2). The reduced
level of NO metabolite excretion observed in the 4a/b
group was observed at three levels of salt intake,
customary intake (around 13073 mEq/day), high
salt intake (around 31076 mEq/day), and under
salt restriction (around 3974 mEq/day) (Figure 2).
Discussion
Three major genetic polymorphisms of the eNOS
gene have been reported as susceptibility genes in a
Table 5 eNOS 4a/b polymorphism and salt sensitivity
b/b n (%) a/b+a/a n (%)
SS 22 (26.8) 17 (38.6)*
SI 23 (28) 9 (20.5)
SR 37 (45.1) 18 (40.9)
Total n 82 44
SS: salt sensitive; SI: intermediate sensitivity to salt; SR: salt
resistance.
eNOS polymorphisms and salt sensitivity
IS Hoffmann et al
237
number of cardiovascular diseases (Table 7). Two of
three polymorphims, the missense Glu298Asp var-
iant and the VNTRs on intron 4, were investigated,
because of their reported stronger associations with
hypertension and cardiovascular diseases (Table 7).
Ethnicity and eNOS polymorphisms
In addition to disease states, the prevalence of a
specific genetic polymorphism is determined by
ethnicity. The prevalence of eNOS polymorphism
has been established for Caucasians, African-Amer-
ican and Japanese populations. However, such
information is not available for Hispanics.
The Glu298Asp is less common in Japanese
(5–10.2%) and African-Americans (10.5–16%), than
in Caucasians (30–37%).9,12,13,23,26,34,35 The frequency of
the T allele in Venezuelans was comparable to that
described for Caucasians, since the T allele was
found in 30% of the Venezuelan subjects studied
(present study).
The frequency of a allele for the VNTRs of intron 4
was also determined by ethnicity. The a allele was
found more frequently in African-Americans
(27–31%) than in Caucasians (16–18%) or Japanese
(10–13%).9,26,34–37 Once again the frequency of a
allele in Venezuelan subjects most closely compared
to that of the Caucasians, since it was found in 18%
of the Venezuelan subjects studied.
Total n These findings further support the important role
that ethnicity plays in determining the prevalence of
39 genetic polymorphisms. The fact that the prevalence
32 of two eNOS polymorphisms compares best with
55
126 that of Caucasians is in support of the major
European ancestry of the Venezuelan population
studied.

Table 6 eNOs 4a/b polymorphism: BP and electrolyte excretion eNOS polymorphisms, NO production
during high and low salt intake and salt sensitivity

b/b a/b Salt sensitivity is characterized by a substantial

increase in BP following a high intake of salt.38 The
Subjects (n) 55 45 exact cause of salt sensitivity remains unknown.
HNa+-SBP (mmHg) 12172 126732* Both genetic and environmental factors interact to
HNa+-DBP 8071.2 8271.8 determine the BP response to changes in salt
UNa+/day 302712 324725
LNa+-SBP 11671.5 11772
intake.1,38–40 Experimental evidence indicates that
LNa+-DBP 7771 7872 NO plays a key role in endothelial health and salt
UNa+/day 3974 3973 sensitivity.41 In genetic models of salt sensitivity and
D SBP (mmHg) 4.870.9 9.071.6* in human subjects, salt sensitivity appears linked to
D DBP (mmHg) 3.070.8 4.471.4
impaired NO production,2–6 an effect reversed by
D-Una+/day 260712 281714
administration of the NO precursor, L-arginine.42,43
UNa+: urinary sodium excretion in mEq/day during a high-sodium Despite numerous evidences supporting the role for
diet (HNa+) and a low-sodium diet (LNa+). NO in salt sensitivity, prior to this work no studies
Systolic and diastolic BP in mmHg. had been conducted on the relationships between
HNa+- SBP and DBP: BP during a high-sodium diet.
LNa+- SBP and DBP: BP during a low-sodium diet. eNOS polymorphisms and salt sensitivity.
D SBP was calculated as SBP during high salt minus SBP during low In our study, we observed that the presence of the
salt. 4a/b polymorphisms was associated with salt sensi-
D DBP was calculated as DBP during high salt minus DBP during low
salt.
tivity and with a reduction in the excretion of NO
D-UNa+/day was calculated as UNa+/day during high salt minus metabolites. Decreases in plasma NO metabolites
UNa+/day during low salt. have also been reported in healthy Japanese carrying

Journal of Human Hypertension

 eNOS polymorphisms and salt sensitivity
IS Hoffmann et al
238
Table 7 Published studies of association between the eNOS Genotyping (Glu298Asp, Intron-4 eNOS4 and 786T/C) and cardiovascular
related outcomes

Glu298Asp eNOS4 (a/b) 786T-C

Positive Negative Positive Negative Positive Negative

association association association association association association

Hypertension Jachymova et al;8 Benjafield and Li et al;26 Benjafield and Kajiyama et al;33
Miyamoto et al;9 Morris;17 Karnoven Pulkkinen et al;27 Morris;17 Shoji Tsujita et al21
Shoji et al;10 et al;18 Kato et al;19 Rodriguez- et al10
Yasujima et al11 Lustberg et al;20 Sparragon et al;28
Tsujita et al21 Uwabo et al29

AMI Hibi et al;12 Fatini et al30 Hibi et al;12 Fatini et al30

Shimasaki et al13 Hwang et al31

IHD Casas et al;14 Chang et al;22 Casas et al14 Hwang et al;31 Colombo et al;15 Casas et al14
Colombo et al;15 Hingorani et al; 23 Via Pulkkinen et al27 Rossi et al;32
Yoshimura et al16 et al;24 Wang et al25 Yoshimura et al16

Salt This study This study

sensitivity

Glu298Asp ¼ Glu298Asp in exon 7; eNOS4(a/b) ¼ 27 bp repeat in intron 4 (a: 4 repeats, b: 5 repeats); 786 T-C in the 5-flanking (promoter)
region;. AMI: acute myocardial infarction, IHD: ischaemic heart disease.

the 4a/b genotype.37 Owing to the existing relation-
ship between reduced NO levels and salt sensitivity
(see above), we propose that the reduced production
of NO observed in subjects harbouring the 4a/b
genotype could be responsible for the greater
sensitivity of their BP to dietary salt. In addition to
salt sensitivity, the 4a/b polymorphism has been
shown to determine the efficacy of nevibolol, a beta-
blocker that in addition increases the production of
NO.44–47 Nebivolol is known to increase NO levels46
possibly by inhibiting NO oxidation.47 Nebivolol
improved left ventricular hypertrophy and endothe-
lial dysfunction in subjects carrying the b/b eNOS
genotype, but failed to do so in those with the 4a/b
genotype.45 The diminished production of NO
reported in subjects with the 4a/b mutation may
prevent the beneficial effects of nebivolol. These
results indicate that subjects carrying the 4a/b
genotype may be at a higher risk of developing
vascular dysfunction, salt sensitivity and poorer
response to drugs that act by increasing NO avail-
ability.
eNOS polymorphisms, cardiovascular disease
and risk factors
The missense Glu298Asp variant has been corre-
lated with increased incidence of coronary spasm,31
coronary heart disease,22 myocardial infarction,12
essential hypertension,9,11 left ventricular hypertro-
phy, carotid atherosclerosis and cerebral infarc-
tion.48 Whereas in other studies, no association
between 298Asp with coronary artery disease,22–25
essential hypertension,17–21 left ventricular hyper-
trophy and carotid atherosclerosis17,35 and cerebro-
vascular disease29,49 was observed.
In order to provide some light to these controver-
sial results, we evaluated the possible existence of
an association between the two more important
eNOS polymorphisms and early cardiovascular risk
factors. We observed that the 298Asp allele was not
associated with BP, albuminuria or with fasting and
postload glucose levels. However, as previously
reported for Anglo-Celtic17 and African-Ameri-
cans,26 in Venezuelans, the Glu298Asp polymorph-
ism was found associated with modest increases in
triglyceride and LDL-cholesterol (present study). It
thus appears that irrespective of ethnicity, the
presence of the Glu298Asp polymorphism carries
an increased prevalence of cardiovascular risk
factors (higher BMI, LDL-cholesterol and triglycer-
ides). However, the increases were modest, and for
the 298Asp carrying two rather than one allele did
not increase the lipid abnormalities.
Polymorphisms of the VNTR on intron 4 of the
eNOS gene have been associated with hyperten-
sion.26–29 However, such an association was not
found in another studies.10,17 In our work, no
association was found between the a allele and the
baseline BP. However, tracking of the 4a allele with
lower HDL- and higher LDL-cholesterol was re-
ported in Anglo-Celtics17 and with higher LDL-
cholesterol in hypertensive African-Americans.26 In
our study, in Venezuelans, the 4a allele was
associated with increased LDL levels. It thus
appears that the presence of the 4a/b polymorphism
carries an increased prevalence of elevated LDL-
cholesterol.
In summary, the prevalence of the Glu298Asp and
of the 4a/b eNOS polymorphism in Venezuelans is
comparable to that reported for Caucasians. The 4a/b
genotype was found associated with reduced NO
production, increased salt sensitivity and increased

Journal of Human Hypertension


levels of LDL-cholesterol. These observations sug-
gest that the eNOS gene locus may be responsible for
variations in the genetic control of plasma and
urinary levels of NO metabolites, which in turn may
favour a state of salt sensitivity. The 298Asp was
found associated with higher triglyceride and LDL-
cholesterol levels. The role of these polymorphisms
in the development and progression of atherosclero-
tic vascular disease needs further work.
Acknowledgements
This study was supported by FONACIT S1-
2001000679, CDCH-06.30.4362.03, CDCH-06-10.42-
14.01, Scientific Initiative of the Millennium No.
BIRF-VE. Sub-project ‘Center for the Detection and
Treatment of Silent Risk Factors for Cardiovascular
and Metabolic Diseases’ to ISH and LCX, and NSU
President’s Faculty Scholarship Award to LCX,
2002.
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