AJH 2004; 17:560 –567
Relation of Endothelial Nitric
Oxide Synthase Gene to Plasma Nitric
Oxide Level, Endothelial Function, and
Blood Pressure in African Americans
Rongling Li, Deborah Lyn, Rigobert Lapu-Bula, Adefisayo Oduwole,
Priscilla Igho-Pemu, Brenda Lankford, Jan Morgan, Sunday Nkemdechi,
Gang Liu, Cheryl Pack, Natalia Silvestrov, Daniel A. von Deutsch,
Qing Song, Imad K. Abukhalaf, and Elizabeth Ofili
Background: The role of eNOS gene polymorphisms
on plasma nitrite or nitrate (NOx) level, endothelial func-
tion, and blood pressure (BP) remains unclear.
Methods: We estimated the relationship of eNOS poly-
morphisms (the T⫺786C in the 5⬘-flanking promoter re-
gion, T⫺786C; 27-bp repeat in intron 4, eNOS4; and
Glu298Asp in exon 7, G894T) with plasma NOx level,
brachial endothelial function assessed by ultrasound mea-
sure of brachial artery flow-mediated dilation (FMD), and
BP in 60 healthy African Americans, 30 men and 30
women aged 18 to 73 years.
Results: Among them, 73.1%, 23.9%, and 3.0% carried
TT, TC, and CC of T⫺786C, respectively, 14.5%, 27.5%,
53.6%, and 1.4% carried aa, ab, bb, and bc of eNOS4
polymorphism, respectively, and 70.4%, 23.9%, and 5.6%
carried GG, GT, and TT of G894T, respectively. G894T
and eNOS4 were observed in linkage disequilibrium.
Mean values of age, plasma NOx, FMD, systolic and
diastolic BPs were not significantly different (P ⬎ .05) by
I
n 1980, it was first discovered that the vascular endo-
thelium released an unstable substance named endo-
thelium-derived relaxing factor (EDRF).1 In 1987 to
1988, Palmer and colleagues reported that nitric oxide (NO)
synthesized from L-arginine by nitric oxide synthase (NOS)
in endothelial cells accounted for the biological activity of
EDRF.2 Two research groups, Janssens et al3 and Marsden et
Received September 17, 2003. First decision November 12, 2003. Ac-
cepted February 9, 2004.
From the Department of Medicine, Clinical Research Center (RL,
BL, JM, SN, EO), Ultrasound Core Laboratory (RL-B, GL, CP), Section
of Cardiology (AO, EO), Section of General Internal Medicine (PI-P),
Department of Biochemistry (DL), Department of Pharmacology and
Toxicology (NS, DAvD, IKA), and Cardiovascular Research Institute
(QS), Morehouse School of Medicine, Atlanta, Georgia.
0895-7061/04/$30.00
doi:10.1016/j.amjhyper.2004.02.013
eNOS polymorphisms. Plasma NOx level was found to be
associated with systolic BP (r ⫽ 0.51, P ⫽ .03), and
diastolic BP (r ⫽ 0.41, P ⫽ .08), but not with FMD, in
individuals with “a” allele of eNOS4 polymorphism after
adjustment for age, body mass index, serum glucose, and
smoking status.
Conclusions: We reveal a positive association be-
tween plasma NOx level and BP in normotensive African
Americans who carry the “a” allele of eNOS4. Because the
frequency of the rare allele “a” is significantly higher in
African Americans than in other ethnic groups, this finding
may provide a clue to understanding the genetic suscepti-
bility to hypertension in African Americans. Am J Hy-
pertens 2004;17:560 –567 © 2004 American Journal of
Hypertension, Ltd.
Key Words: endothelial nitric oxide synthase gene,
nitric oxide, endothelial function, blood pressure, African
Americans.
al,4 isolated the cyclic DNA for the human vascular endo-
thelial NOS in 1992. Thereupon, the endothelial nitric oxide
synthase (eNOS or NOS3) gene was mapped on chromo-
some 7q36.5 The eNOS gene consists of 26 exons spanning
approximately 21 kb of genomic DNA and encoding an
mRNA of 4052 nucleotides.
Studies using cell culture and animal model revealed
This study was supported by grants U54RR14758-01, U54RR14758-
02, U54RR14758-03, and CRC 2P20RR1104 from the National Insti-
tutes of Health, Bethesda, Maryland.
Address correspondence and reprint requests to Dr. Rongling Li,
University of Tennessee, Health Science Center, Department of Preven-
tive Medicine, 66 N. Pauline, Suite 633, Memphis, TN 38163; e-mail:
rli2@utmem.edu
© 2004 by the American Journal of Hypertension, Ltd.
Published by Elsevier Inc.
AJH–July 2004 –VOL. 17, NO. 7 eNOS GENES, PLASMA NOx , ENDOTHELIAL FUNCTION, AND BP
that free radical NO is important in the regulation of
vasomotor tone and blood flow by inhibiting smooth mus-
cle contraction6,7 and platelet aggregation.8 This NO is
scavenged rapidly and acts in a paracrine fashion to trans-
duce cellular signals. Plasma nitrite (NO2⫺) or nitrate
(NO3⫺) are stable oxidation products of free radical NO.9
However, previous reports on the relationship of eNOS
gene to plasma nitrite (NO2⫺) and nitrate (NO3⫺) lev-
els,10,11 blood pressure (BP),12,13 or heart disease14,15 in
human are controversial. Few studies reported the rela-
tionship of the eNOS genotypes to plasma NO products
and clinical phenotypes in African Americans. In this
study, we assessed the relationship of the selected eNOS
polymorphisms of T⫺786C, a single nucleotide polymor-
phism with T to C mutation in the promoter region, 27-bp
repeat in intron 4 (eNOS4) and Glu298Asp (G894T) in
exon 7 to plasma NOx, brachial endothelial function as-
sessed by ultrasound measure of brachial flow-mediated
dilation (FMD), and BP in normotesive African Ameri-
cans. We also assessed the association between plasma
NOx and BP, as well as plasma NOx and brachial endo-
thelial function separately for different genotypes.
Methods
Participants and Data Collection
The study participants were recruited from shopping
malls, barbershops, community centers, and health fairs in
the south and southwest areas of metropolitan Atlanta.
They were self-reported African Americans aged 18 years
or older who did not have physician-diagnosed high BP,
diabetes, coronary heart disease, stroke, or any life-threat-
ening diseases, and who had no history of drug abuse.
Participants’ eligibility was further confirmed by question-
naire and clinical examinations after enrollment in the
study. The study enrolled 72 participants (38 women and
34 men). We excluded 7 subjects whose BP (systolic
BP/diastolic BP ⱖ140/90 mm Hg) met the definition of
hypertension, as reported by the Sixth Joint National Com-
mittee on Prevention, Detection, Evaluation and Treat-
ment of High Blood Pressure, and excluded 5 individuals
with incomplete laboratory assays. The final participants
in this study were 30 female and 30 male African Amer-
ican normotensives.
Individuals who agreed to participate in the study were
invited to the Morehouse School of Medicine (MSM)
Clinical Research Center (CRC) for interview and clinical
examinations. The details of the study and informed con-
sent were fully explained to all participants by the study
coordinator. Before the enrollment, participants signed the
consent form approved by the MSM Institutional Review
Board. Thereafter, an interviewer-administered question-
naire was applied to collect participants’ demographic,
lifestyle, socioeconomic information, as well as personal
medical history and family history of hypertension, diabe-
tes, coronary heart disease, or stroke. A physician con-
ducted the physical examination. A trained nurse
561
conducted participants’ height, weight, BP measurements,
and electrocardiographic examination. Participant’s BP
was measured in the supine position after 15 min of rest.
Overnight fasting blood (30 mL) was drawn and processed
(transferred to different tubes, separated by centrifugation,
frozen, and packaged in ⫺70°C freezer) by a trained
technician at the CRC laboratory. Blood samples were sent
to the MSM molecular genetics laboratory for genotype
assays, to the MSM analytical laboratory for quantitation
of plasma NOx, and to Doctors Laboratory, Inc (Valdosta,
GA) for routine laboratory tests of metabolic panel, lipid
profile, and hematology. All blood samples were labeled
with participants’ identification numbers. Laboratory per-
sonnel were blind to participants’ information.
Genotype Assay of eNOS
Genomic DNA was extracted from 1 mL of whole blood
using the QIAamp DNA blood kit (Qiagen, Valencia,
CA). Genotyping of each polymorphism was performed
by amplification from 30 to 50 ng of genomic DNA. The
eNOS polymorphisms selected in this study were
T⫺786C in the promoter region, eNOS4 in intron 4, and
G894T in exon 7 (see Fig. 1 for their locations). The
eNOS 4a/4b genotype was determined using primers
that flank the 27-bp direct repeat using the following
primers: (forward) 5⬘-AGGCCCTATGGTAGTGC-
CTT-3⬘ and (reverse) 5⬘-TCTCTTAGTGCTGTGGT-
CAC-3⬘. Primers for the G894T and T⫺786C
polymorphisms were as described by Tanus-Santos et
al.16 Each assay was conducted in 25 ␮L containing 1.0
U Taq DNA polymerase (Qiagen), 0.5 ␮mol/L of the
appropriate primer, and 250 ␮mol/L of each dNTP. The
reaction for amplifying the T⫺786C genotype contained
2% dimethyl sulfoxide. Each reaction was initially de-
natured for 1 min at 94°C. This step was followed by 34
cycles at 95°C for 25 sec, 56°C (4a4b and Glu298Asp)
or 62°C (T⫺786C) for 35 sec, 72°C for 40 sec, with a
final extension at 72°C for 5 min.
The eNOS alleles 4a, 4b, and 4c were identified as
fragments corresponding to 393-bp, 420-bp, and 447-bp,
respectively, after separation on 4% NuSieve 3:1 agarose
gel (FMC Bioproducts, Rockland, ME) stained with
ethidium bromide (Fig. 2). In addition, the 27-bp repeat
elements were verified by DNA sequencing. Amplified
FIG 1. The human eNOS gene and locations of genetic polymor-
phisms. The eNOS has 26 exons (gray boxes). Introns that contain
polymorphisms are indicated by an asterisk. The gray daggers
indicate genetic variants in exons and intron. Six SNPs in the 5⬘-
flanking region and one SNP in the 3⬘-flanking region are shown as
an asterisk.
562 eNOS GENES, PLASMA NOx , ENDOTHELIAL FUNCTION, AND BP
FIG 2. Allele-specific banding patterns of eNOS 27-bp repeat in
intron 4.
products of the G894T genotype were incubated with
BanII or MboI restriction enzyme, whereas the T⫺786C
genotypes were digested with MspI before gel analysis as
described above.
Quantitation of Plasma NOx
Concentrations for the nitric oxide products, total nitrate/
nitrite, were determined colorimetrically by using an assay
kit from the Cayman Chemical Company (Ann Arbor,
MI). Briefly, plasma samples (40 ␮L) were incubated at
room temperature for 3 hours with nitrate reductase to
convert nitrates into nitrites. Greiss reagent (50 ␮L) was
then added to convert nitrite into a chromophore. Absor-
bance was measured spectrophotometrically at 540 nm
using a Spectramax Plus Microplate Reader (Molecular
Device, Sunnyvale, CA). The concentration of nitric oxide
products (in micromoles per liter) were calculated as total
nitrate ⫹ nitrite from a nitrate standard curve. Each plasma
sample was run in triplicate and the average was reported
as the final plasma NOx level. To assess measurement
variability, approximately 6% of the samples from this
study and our other ongoing studies were replicates un-
known to laboratory personnel. A correlation coefficient of
0.96 between samples and their replicates indicated a very
good reproducibility of this measurement.
Determination of
Brachial Endothelial Function
Brachial endothelial function was determined by brachial
artery vasodilator response or brachial FMD. It was mea-
sured in the dominant arm according to previously vali-
dated techniques.17 An 8.0-MHz linear phased array
ultrasound transducer attached to an ATL5000 (Philips
Medical Systems, Bothell, WA) was used to perform di-
ameter measurements and Doppler flow parameters of the
brachial artery. Participants were imaged in the supine
position. After baseline measurements of brachial artery
diameter and Doppler spectral velocity, a BP cuff was
inflated on the proximal portion of the arm to 200 mm Hg
for 5 min, creating distal limb ischemia. After release of
the cuff, reactive hyperemia occurred (flow-mediated or
endothelium-dependent vasodilation). The brachial artery
was imaged continuously for 2 min after cuff release and
maximum diameter selected. Maximum Doppler flow ve-
AJH–July 2004 –VOL. 17, NO. 7
locity was similarly recorded for reactive hyperemia. Bra-
chial flow-mediated vasodilation was calculated as (HD ⫺
BD)/BD), where HD refers to the hyperemia diameter and
BD is the baseline diameter. Studies in our laboratory have
confirmed the reproducibility of this technique in healthy
volunteers, with intra- and interobserver variability of less
than 10%.
Statistical Analysis
The SAS/Genetics version 9.0 (SAS Institute Inc., Cary,
NC) was used to test Hardy-Weinberg equilibrium and
linkage disequilibrium, to measure marker informative-
ness, and to estimate the haplotype frequency of T⫺786C,
eNOS4, and G894T of eNOS polymorphisms with un-
known gametic phase. Hardy-Weinberg equilibrium was
tested by permutation version of exact test with a modified
version of the Markov-chain random walk algorithm.
Linkage disequilibrium between a pair of loci was tested
using Weir’s ␹2 statistic and a likelihood-ratio test. The
allele association between a pair of loci was measured by
the correlation coefficient (r) and Lewontin’s D⬘. Marker
informativeness was measured by polymorphism informa-
tion content (PIC), heterozygosity and allelic diversity.
Maximum-likelihood estimates of haplotype frequency
were computed using an expectation-maximization (EM)
algorithm. The SAS/STAT was used to compute mean and
95% confidence interval of mean for the variables of
interest. The significant difference of means by the geno-
type of the selected eNOS polymorphisms was determined
by analysis of covariance with adjustment for age, sex,
body mass index (BMI), serum glucose, and smoking
status. Pearson’s correlation analysis was used to estimate
correlations between NOx and BP, NOx and FMD, and BP
and FMD overall and stratified by genotype. To control for
confounders, we estimated partial correlation coefficients
controlling for age, smoking, BMI, and glucose, the fac-
tors potentially related to BP, endothelial function, and
plasma NOx.18,19 Haplotype trend regression was applied
to assess the association of all estimated haplotyes with
NOx, FMD, systolic and diastolic BP.20
Results
Distribution of the
Selected eNOS Polymorphisms
The distribution of T⫺786C and G894T in the sample of 60
African Americans did not deviate from the Hardy-Wein-
berg equilibrium. The allele frequencies of the eNOS
polymorphisms and three measures of allele informative-
ness (PIC, heterozygosity, and allele diversity) are shown
in Table 1. The alleles T of T⫺786C, b of eNOS4, and G of
G894T are common alleles with frequency greater than
60% in this sample. The higher values of allele informa-
tiveness for eNOS4 comparing with that of T⫺786C and
G894T suggested a higher allelic variation of eNOS4. This
high allelic variation tends to be more informative and
AJH–July 2004 –VOL. 17, NO. 7 eNOS GENES, PLASMA NOx , ENDOTHELIAL FUNCTION, AND BP
Table 1. Allele frequency and 95% confidence interval (CI) of selected eNOS polymorphisms
Locus Allele Frequency 95% CI
T786C C 0.167 0.100–0.242
T 0.833 0.758–0.900
eNOS4 a 0.292 0.208–0.383
b 0.692 0.600–0.775
c 0.017 0.000–0.042
G894T G 0.850 0.775–0.917
T 0.150 0.083–0.225
T⫺786C in the 5⬘-flanking (promoter) region; eNOS4(a/b/c) ⫽ 27-bp repeat in intron 4 (a ⫽4 repeats, b ⫽5 repeats, c ⫽6 repeats); G894T ⫽
Glu298Asp in exon 7.
more useful in association studies. The genotype frequen-
cies were 73.1%, 23.9%, and 3.0% for TT, TC, and CC of
T⫺786C, respectively, 14.5%, 27.5%, 53.6%, and 1.4% for
aa, ab, bb, and bc of eNOS4, respectively, and 70.4%,
23.9%, and 5.6% for GG, GT, and TT of G894T, respec-
tively. Maximum-likelihood estimates of haplotype fre-
quency of eNOS T⫺786C, eNOS4, and G894T
polymorphisms were 6.0% of CaG, 23.3% of TaG, 5.4%
of CbG, 49.2% of TbG, 1.6% of TcG, 2.6% of CbT, and
11.1% of TbT. As expected, the frequencies of genotype
and haplotype were not significantly different by gender
(data not shown). Linkage disequilibrium test for all pairs
of loci indicated an association between eNOS4 and
G894T. This is consistent with the report from a previous
study.16 The correlation coefficients (r) between eNOS4
alleles and G894T alleles were 0.33 of a-G, ⫺0.33 of a-T,
⫺0.36 of b-G, 0.36 of b-T, 0.11 of c-G, and ⫺0.11 of c-T.
Association of eNOS
Genotype/Haplotype With
Plasma NOx, Flow-Mediated Dilation,
BP, and Cardiovascular Risk Factors
The distributions of plasma NOx, FMD, BP, and selected
cardiovascular risk factors by different eNOS genotypes
are listed in Table 2. We combined the homozygous bb
with heterozygous bc (n ⫽ 2) of eNOS4, TC with CC (n
⫽ 2) of T-784C, and GT with TT (n ⫽ 2) of G894T,
because the number of rare alleles or genotypes was too
small to make comparison with others. Mean values of
plasma NOx, FMD, BP, and selected cardiovascular risk
factors were not significantly different by genotypes before
(Table 2) and after (data not shown) adjustment for age,
sex, BMI, serum glucose, and smoking status. In this
sample, there were 25.4% smokers (6.8% current smokers
and 18.6% former smokers). The proportions of ever
smoking did not significantly differ by genotype (Table 2,
similar results, data did not show, for current and former
smoking). In the haplotype trend regression, in which the
estimated probability of possible haplotype for each indi-
vidual was the predictor, no association of any haplotype
with NOx, FMD, systolic and diastolic BP was observed
before or after adjustment for the selected covariates.
563
PIC Heterozygosity Allele Diversity
0.24 0.27 0.28
0.35 0.32 0.44
0.22 0.23 0.26
Correlation Between
Flow-Mediated Dilation and BPs
Overall, FMD was inversely but not significantly corre-
lated with systolic (r ⫽ ⫺0.22, P ⫽ .08) and diastolic (r
⫽ ⫺0.17, P ⫽ .20) BPs among the sample of normoten-
sive African Americans. After adjusted for age, BMI,
serum glucose, and cigarette smoking, the correlation co-
efficients decreased, r ⫽ ⫺0.05, P ⫽ .71 for FMD corre-
lated with systolic, and r ⫽ ⫺0.06, P ⫽ .70 for FMD
correlated with diastolic BP. None of the correlation co-
efficients between FMD and BP were a statistically sig-
nificant departure from zero after stratified by each
genotype of the three eNOS polymorphisms.
Relationship of Plasma NOx
With Flow-Mediated Dilation and BP
The plasma NOx level was not correlated with FMD
overall or stratified by the genotype of the selected eNOS
polymorphisms. No association between the plasma NOx
level and BP was observed in the 60 African Americans as
a whole. Interestingly, when the assessment of the plasma
NOx level and BP association was stratified by the geno-
type of these eNOS polymorphisms, the plasma NOx level
was positively correlated with BP, especially with systolic
BP in the participants who carried the a allele in 27-bp
repeat of intron 4 (Table 3 and Fig. 3). The correlation
coefficients in a allele carriers were r ⫽ 0.51 (P ⫽ .03) for
plasma NOx and systolic BP, and r ⫽ 0.41 (P ⫽ .08) for
plasma NOx and diastolic BP after adjustment for age, sex,
BMI, glucose, and cigarette smoking. This correlation was
not observed in those who were bb homozygotes of 27-bp
repeat in intron 4. There was no relationship between
plasma NOx level and BP observed in different genotypes
of T⫺786C and G894T.
Discussion
The genotype distribution of T⫺786C in this study was
similar to a previous report in unrelated African Ameri-
cans of undetermined clinical status.16 However, the fre-
quencies of the rare allele TT homozygotes (5.6%) of
G894T and aa homozygotes (14.5%) of the 27-bp repeat in
 564
eNOS GENES, PLASMA NOx , ENDOTHELIAL FUNCTION, AND BP
Table 2. Mean and 95% confidence interval (CI) of selected factors by eNOS genotype

T786C(TT) T786C(TC/CC) eNOS4(aa) eNOS4(ab) eNOS4(bb/bc) G894T(GG) G894T(GT/TT)

(n ⴝ 42) (n ⴝ 18) (n ⴝ 9) (n ⴝ 17) (n ⴝ 34) (n ⴝ 44) (n ⴝ 16)
9.5 8.1 9.5 8.1 9.5 8.8 9.9
NOx (␮M) (8.3, 10.8) (6.9, 9.3) (7.0, 12.0) (6.2, 10.0) (8.3, 10.8) (7.7, 9.9) (8.1, 11.7)
14.5 15.8 13.7 16.1 14.6 14.7 15.6
FMD (%) (13.3, 15.8) (12.5, 19.1) (12.4, 14.9) (13.0, 19.3) (13.0, 16.3) (13.2, 16.1) (12.6, 18.7)
119.5 118.0 120.4 118.6 118.9 118.8 119.6
SBP (mm Hg) (116.2, 122.7) (1118.0, 124.2) (112.5, 128.3) (115.0, 122.3) (114.4, 123.3) (115.4, 122.2) (113.9, 125.3)
73.4 77.1 74.9 75.4 74.0 73.7 76.8
DBP (mm Hg) (70.2, 76.5) (73.0, 81.2) (67.6, 82.2) (72.0, 78.7) (70.0, 77.9) (70.9, 76.5) (70.8, 82.7)
39.2 41.5 44.7 38.1 39.6 38.2 44.5
Age (yr) (34.9, 43.5) (35.6, 47.4) (38.7, 50.7) (30.6, 45.5) (34.8, 44.3) (34.0, 42.4) (39.1, 49.9)
28.5 26.1 26.4 26.8 28.6 27.0 30.0
BMI (kg/m2) (26.0, 31.0) (24.6, 27.7) (22.7, 30.1) (22.7, 31.0) (26.2, 31.1) (25.1, 28.8) (25.2, 34.9)
95.7 89.3 87.9 86.4 99.4 92.6 97.1
Glucose (mg/dL) (87.1, 104.2) (84.7, 94.0) (81.7, 94.1) (82.0, 90.7) (88.7, 110.0) (88.0, 97.3) (76.7, 117.4)
123.9 138.9 134.4 143.6 119.4 123.9 137.1
LDL (mg/dL) (110.0, 137.8) (116.7, 161.0) (98.4, 170.4) (122.3, 164.9) (103.9, 134.9) (108.4, 139.3) (121.3, 152.8)
49.3 54.0 47.8 52.1 50.5 50.5 51.1
HDL (mg/dL) (45.0, 53.5) (45.1, 62.9) (33.8, 61.8) (42.9, 61.2) (45.8, 55.1) (45.5, 55.5) (44.7, 57.6)
112.7 131.9 103.6 94.9 132.6 113.3 130.6
Triglyceride (mg/dL) (86.8, 138.6) (86.0, 177.7) (74.5, 132.7) (78.7, 111.2) (97.2, 168.1) (88.9, 137.7) (79.9, 181.2)
Smoking (%)* 26.8 22.2 33.3 25.0 23.5 20.5 40.0

BMI ⫽ body mass index; DBP ⫽ diastolic blood pressure; eNOS ⫽ endothelial nitric oxide synthase; eNOS polymorphisms of T⫺786C in the 5⬘-flanking (promoter) region; eNOS4(a/b/c) ⫽ 27-bp
repeat in intron 4 (a ⫽4 repeats, b ⫽5 repeats, c ⫽6 repeats); FMD ⫽ brachial flow-mediated dilation; G894T ⫽ Glu298Asp in exon 7; Nox ⫽ plasma nitrite/nitrate; PBP ⫽ pulse blood pressure is
SBP–DBP; SBP ⫽ systolic blood pressure.

AJH–July 2004 –VOL. 17, NO. 7

* Smoking: ever versus never, no statistically significant difference by genotypes of each polymorphism.
AJH–July 2004 –VOL. 17, NO. 7 eNOS GENES, PLASMA NOx , ENDOTHELIAL FUNCTION, AND BP 565

Table 3. Relationship of plasma nitrite/nitrate with blood pressure and FMD by eNOS4a/b/c genotypes

SBP DBP FMD

Adjusted
n r P r P r P for
eNOS4(aa/ab) 26 0.37 0.07 0.40 0.04 ⫺0.23 0.27
0.39 0.05 0.47 0.02 ⫺0.24 0.25 Age
0.47 0.02 0.40 0.05 ⫺0.10 0.62 BMI
0.38 0.06 0.39 0.06 ⫺0.22 0.29 Glucose
0.37 0.09 0.34 0.12 ⫺0.20 0.36 Smoking
0.51 0.03 0.41 0.08 ⫺0.04 0.86 All
eNOS4(bb/bc) 34 ⫺0.05 0.77 0.04 0.81 0.05 0.80
⫺0.08 0.68 ⫺0.10 0.57 0.04 0.83 Age
⫺0.08 0.68 ⫺0.06 0.73 0.06 0.74 BMI
⫺0.01 0.96 0.02 0.94 0.03 0.87 Glucose
⫺0.03 0.88 ⫺0.003 0.98 0.04 0.84 Smoking
⫺0.001 0.99 ⫺0.05 0.81 0.10 0.63 All

eNOS4(aa/ab) ⫽ the combination of aa homozygous and ab heterozygous; eNOS4(bb/bc) ⫽ bc heterozygous and bb homozygous of 27-bp
repeat in intron 4; other abbreviations as in Table 2.

intron 4 were higher in this study compared to the previous
study (TT: 1.0%, aa: 6.0%).16 The frequency of bb ho-
mozygotes (53.3%) of the 27-bp repeat in intron 4 in our
study was also higher than that in the previous report
(45.0%).16 Distribution of all polymorphisms is under the
FIG 3. Relationship of plasma nitric oxide (NO) with (A) systolic
blood pressure (SBP), (B) diastolic blood pressure (DBP), and (C)
brachial flow-mediated dilation (FMD) among 26 African American
normotensives with eNOS (4aa or ab) genotypes.
Hardy-Weinberg equilibrium, which suggests the results
of this study are unlikely to be biased by population
stratification or admixture.
In this study, none of the analyzed eNOS polymor-
phisms or their haplotypes was significantly associated
with plasma NOx level, although Tsukada et al10 reported
high plasma NOx levels in healthy Japanese who were
homozygous bb carriers, whereas Wang et al11 reported a
contradictory result of high plasma NOx levels in healthy
volunteers of European descendant who were homozygous
aa of the 27-bp repeat in intron 4.
Reports on the relationship of eNOS gene variants to
BP alterations are controversial.12,13,21 In this study of
normotensive African Americans, we did not observe any
relationship of the selected eNOS polymorphisms or pos-
sible haplotype with BP level, or with brachial endothelial
function assessed by FMD before and after adjustment for
age, sex, BMI, serum glucose, and cigarette smoking.
However, this study showed increased BP, especially sys-
tolic BP, with increased plasma NOx levels only in a allele
carriers of the 27-bp repeat in intron 4 after adjustment for
age, sex, BMI, serum glucose, and cigarette smoking, the
factors known to be related to both plasma NOx level and
BP. The a allele of the 27-bp repeat in intron 4 is a rare
allele, but the frequency of a allele and homozygotes aa
was found to be significantly higher in African Americans
compared with other racial/ethnic groups.16 Moreover, this
rare allele 4a associated with common allele G of G894T
in exon 7 (r ⫽ 0.3, Lewontin’s D⬘ ⫽ 0.62) may be a
biomarker for BP variation in African Americans. In fact,
previous studies observed a relationship of the 4a variant
with cardiovascular and renal diseases in African Ameri-
cans.14,22
Studies of eNOS gene knockout mouse indicated that
lack of eNOS, the enzyme for NO synthesized from L-
arginine, was associated with increased BP.23 Overexpres-
sion of eNOS has been found to be associated with
566 eNOS GENES, PLASMA NOx , ENDOTHELIAL FUNCTION, AND BP
hypotension.24 –26 Plasma NOx is the oxidation product of
free radical NO.9 Its concentration may reflect the free
radical NO level in endothelial cell.27 Thus, the positive
association between plasma NOx and BP in our study of
normotensive African Americans carrying the a allele of
eNOS4 appears counterintuitive to previous studies. How-
ever, recent studies found that the chronic overexpression
of eNOS in the endothelium resulted in resistance to the
NO/cyclic GMP-mediated vasodilators, and that at least
two distinct mechanisms might be involved: one is re-
duced soluble guanylate cyclase (sGC) activity, and the
other is a decrease in cyclic GMP-dependent protein ki-
nase (PKG) protein levels.28,29 Therefore, we speculate
that the increased BP with increase plasma NOx levels in
a allele of eNOS4 carriers may be explained by chronic
overexpression of eNOS in this subgroup.
Apart from the genetics for eNOS, the corresponding
increase in systolic BP with NOx might be the result of a
complex interaction with free radicals, inactivating vascu-
lar relaxing effects of NO. Nitric oxide is a weak radical
with a relatively long half-life that scavenges more highly
reactive free radicals such as superoxides (O2⫺) produced,
for example, by the enzyme NADP(H) oxidase.30 The
interaction of superoxides with NO can result in the gen-
eration of reactive nitrogen species including peroxyni-
trites, nitrites, and nitrates. Furthermore, evidence
indicates that an imbalance between the antioxidant effects
of NO and vascular oxidative stress can result in vascular
dysfunction.31 Thus, if NO is forced to act as an antioxi-
dant rather than as a vasorelaxant, its efficacy would be
attenuated. However, whether this process is contributing
the observed variability associated with plasma NOx levels
and systolic BP remains to be elucidated.
Plasma NOx level can also be influenced by adminis-
tration of exogenous pharmacologic sources of NO, diet,
or bacterial infection. In this study, no one was under
nitroglycerin or cardiovascular disease treatment, which
ruled out the pharmacologic influence. Although we were
not able to control for the diet of all participants before
they visited the Clinical Research Center, 15% of them
had 1-week low/high salt diet at the Clinical Research
Center for cardiac functional study. The correlation of
plasma NOx level before and after their 7-day diet control
was 0.74, which indicated diet effects on plasma NOx were
minor in our study. In addition, the genotyping and NOx
assay (phenotyping) were conducted independently by dif-
ferent technicians in different laboratories without know-
ing results from each other. Diet effects on the plasma NOx
levels, if any, should be random across the eNOS genotype
groups, unless individuals with specific variants had a
predisposition to consume a diet that significantly altered
their plasma NOx levels. Cigarette smoking was reported
to affect both the endothelial function32,33 and plasma NOx
level.34 Yoon and colleagues34 found a significantly higher
concentration of plasma NOx in ever-smokers (24.4%
current and 6.9% former smokers) than nonsmokers, es-
pecially in a allele of eNOS4 carriers. However, in our
AJH–July 2004 –VOL. 17, NO. 7
study, we did not find any significant difference of plasma
NOx by smoking and eNOS4 genotype. Plasma NOx levels
(95% confidence interval) were 7.7 ␮mol/L (4.9, 10.4) of
ever-smokers and 9.1 ␮mol/L (7.2, 11.0) of nonsmokers in
aa/ab of eNOS4 carriers, and were 9.2 ␮mol/L (5.2, 13.2)
of ever-smokers and 9.6 ␮mol/L (8.3, 11.0) of nonsmokers
in bb/bc carriers. This inconsistence between our and
Yoon’s study may be due to the small proportion (6.8%) of
current smokers in our sample with a limited power to
detect the effect, if increased plasma NOx levels in smok-
ers result mainly from current smoking.
This study had a small number of participants (n ⫽ 60)
after excluding ineligible individuals and those with miss-
ing data, which precluded the ability to detect weak asso-
ciations. Some negative results such as no association
between eNOS4 and plasma NOx or BP may be due to
insufficient statistical power compared to other reports.
The positive result of plasma NOx associated with BP in a
allele of eNOS4 carriers (n ⫽ 26) might also be chance-
related (false positive). However, as both plasma NOx and
BP were continuous measures, which increase statistical
power, with no significant outlier (Fig. 3) in this sample,
the result should be able to provide a clue for future
studies.
In conclusion, we found a positive correlation between
BP and plasma NOx in individuals with the rare allele a of
eNOS 27-bp repeats in intron 4. Future studies in African
Americans with a larger sample size are needed to confirm
this result.
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