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ORIGINAL ARTICLE
Diagnostic value of nitric oxide, lipoprotein(a), and
malondialdehyde levels in the peripheral venous and cavernous
blood of diabetics with erectile dysfunction
MA El-Latif
1
, AA Makhlouf
2
, YM Moustafa
3
, TE Gouda
1
, CS Niederberger
2
and SM Elhanbly
3
1
Medical Biochemistry Department, Mansoura University, Mansoura, Egypt;
2
Department of Urology, University of
Illinois, Chicago, IL, USA and
3
Andrology Department, Mansoura University, Mansoura, Egypt
Diabetes mellitus (DM) is the single most common cause of erectile dysfunction (ED) seen in clinical
practice. Evaluation of penile arterial insufficiency in diabetic patients currently entails expensive
and invasive testing. We assessed the diagnostic value of certain peripheral and cavernous blood
markers as predictors of penile arterial insufficiency in diabetic men with ED. This study was
conducted on a total of 51 subjects in three groups: 26 impotent diabetics, 15 psychogenic impotent
men and 10 normal age matched control males. All subjects underwent standard ED evaluation
including estimation of postprandial blood sugar and serum lipid profile. Peripheral venous levels
of nitric oxide (NO), lipoprotein(a) (LP(a)), malondialdehyde (MDA) and glycosylated hemoglobin
(HbA1c) were obtained in all subjects. Patients in the two impotent groups underwent additional
measurement of NO, LP(a) and MDA levels in cavernous blood. They also underwent
intracavernosal injection (ICI) of a trimix (papaverine, prostaglandin E1 and phentolamine
mixture) and pharmaco-penile duplex ultrasonography (PPDU). Compared to patients in the
psychogenic group, diabetic men had significantly lower erectile response to ICI (Po0.001), lower
peak systolic velocity (PSV) (Po0.001), and smaller increase in cavernosal artery diameter (CAD)
(Po0.001). Peripheral and cavernous levels of both LP(a) and MDA were higher in the diabetic
group as compared to the psychogenic ED group (Po0.001), while the values of peripheral venous
and cavernous NO were lower (Po0.001) in the diabetic men. Comparison of biochemical marker
assays with the PPDU results showed a significant negative correlation between both venous and
cavernous LP(a) and MDA levels on the one hand, and PSV, and the percentage of CAD increase on
the other. At the same time, peripheral and cavernous NO levels had a significant positive
correlation with the same parameters. Lipoprotein(a), MDA and NO levels were better predictors of
low PSV than HbA1c, cholesterol or triglyceride levels. The finding of high levels of LP(a) and MDA
with low levels of NO in the peripheral and cavernous venous blood of diabetic men with ED
correlates strongly with severity of ED as measured by PPDU. This provides a rationale for further
studies of biochemical markers as a surrogate for traditional invasive testing in the diagnosis of
penile arterial insufficiency.
International Journal of Impotence Research (2006) 18, 544–549. doi:10.1038/sj.ijir.3901473;
published online 20 April 2006
Keywords: diabetes; impotence; nitric oxide; lipoprotein; malondialdehyde
Introduction
Erectile dysfunction (ED) is frequently associated
with diabetes mellitus (DM).
1,2
Diabetic impotence
is thought to be mainly due to a combination of
vascular and neurogenic changes.
3
It is becoming
increasingly evident that endothelial and smooth
muscle functions are disordered in diabetes and
these may be the most important factor in the
development of ED in a majority of diabetic men.
4,5
Nitric oxide (NO) has been the subject of intense
interest since its biological description in 1987.
6
It
is synthesized from L-arginine by a family of nitric
oxide synthetases (NOS). The release of NO by
cholinergic and non-cholinergic nerves appears to
be the major contribution of the parasympathetic
Received 17 January 2006; revised 3 March 2006; accepted
6 March 2006; published online 20 April 2006
Correspondence: Dr C Niederberger, Department of Urol-
ogy, University of Illinois, 820 S Wood St, MC 955,
Chicago, IL 60612-7316, USA.
E-mail: craign@uic.edu
International Journal of Impotence Research (2006) 18, 544–549
&
2006 Nature Publishing Group All rights reserved 0955-9930/06 $30.00
www.nature.com/ijir
nervous system during erection. Nitric oxide is now
understood to be the most significant mediator of
vascular smooth muscle relaxation responsible for
engorgement of erectile tissue in men.
7–9
Hypercholesterolemia in diabetic patients is a
contributing factor in atherosclerosis and also leads
to an increased risk of impotence.
2
Lipoprotein(a)
(LP(a)) is a glycoprotein component of low-density
lipoprotein (LDL) which is attached to apolipopro-
tein B-100. Lipoprotein(a) may enhance fibrous
plaque formation in atherosclerotic lesions, and its
elevation has been suggested to be an independent
risk factor for atherosclerosis.
10
Oxidative stress is believed to affect the develop-
ment of diabetic-associated vasculopathy, endothe-
lial dysfunction and neuropathy within erectile
tissue.
11,12
Malondialdehyde (MDA) is a known by-
product of reactive oxygen species (ROS) metabo-
lism.
13
Griesmacher et al.
14
observed a significant
rise in MDA in diabetic patients. With increased
peroxidation and reduced antioxidant reserve in
diabetes, oxidative stress may play an important
role in the pathogenesis of diabetic vascular compli-
cations.
13
The aim of this work was to study the plasma
levels of NO, LP(a) and MDA in peripheral and
cavernous blood of diabetic ED patients, and to
correlate these biochemical parameters with other
invasive procedures for the diagnosis of penile
arterial insufficiency.
Subjects and methods
Subject groups
This study included 26 type II diabetic men with
ED, with ages ranging from 33 to 60 years (mean
44.975.9 years) and a mean duration since diag-
nosis of diabetes of 3.3 years. Of the 26 patients, 12
were on insulin therapy. Fifteen impotent patients
with psychogenic etiology with ages ranging from
32 to 59 years (mean 42.878.4 years) were enrolled
for comparison. Finally, 10 normal potent men with
the same age range (mean 40.375.1 years) were
taken as a healthy control group. Informed consent
was obtained from all patients.
We excluded diabetic patients with macro-vascular
complications (coronary artery disease, hyperten-
sion, transient ischemic attacks, strokes, peripheral
ischemia) and overt peripheral neuropathy. We also
excluded patients using antihypertensive drugs,
systemic steroids, histamine-2 blockers and anti-
depressants. All cigarette smokers and alcoholics
were also excluded.
Intracavernosal injection
Intracavernosal injection (ICI) was carried out for
the two impotent groups only. Injections consisted
of 0.5 ml of a Trimix solution containing 30 mg/ml of
papaverine hydrochloride, 10 mg/ml of prostaglan-
din E1 and 1 mg/ml of phentolamine. Patients were
then observed for 1 h to assess the erectile response
by a physician who was blinded to the results of the
biochemical assays.
Pharmaco-penile duplex ultrasonography
Pharmaco-penile duplex ultrasonography (PPDU)
was performed, again, for the two impotent groups
only. It included measurements of cavernosal artery
diameter (CAD) before and after ICI, peak systolic
velocity (PSV) and end diastolic velocity (EDV)
using a 7.5 MHz Toshiba SAL 270-A transducer
(Toshiba Corp., Japan). Measurements of CAD and
blood flow velocities were repeated approximately
2, 5, 10 and 20 min after the injection of Trimix.
Biochemical investigations
Fasting blood samples were taken from the ante-
cubital vein of all impotent patients and all healthy
controls. An additional sample was taken from the
flaccid penis of impotent diabetic and psychogenic
patients only. All blood samples were collected in
tubes containing EDTA, centrifuged and plasma was
separated and divided into aliquots, which were
stored at 201C till assayed. One part of the whole
peripheral venous blood was used for the determi-
nation of HbA1c by ion exchange resin procedure
15
using kits supplied by Stanbio, USA.
Plasma NO assay involved the conversion of
nitrate to nitrite by the enzyme nitrate reductase.
The detection of total nitrite was determined as a
colored azodye product of the Griess reaction that
absorbs visible light using a micro plate reader set
at 540 nm. According to the method of Schmidt
and Walter,
16
nitrate reductase enzyme was used
with nicotinamide adenine dinucleotide (NADH) as
cofactor to reduce nitrate to nitrite. This assay was
performed using kits supplied by R&D Systems Inc.
(Minneapolis, MN, USA).
For the MDA assay, the lipid peroxide content in
plasma was determined by measuring the thiobarbi-
turic acid-reactive substances expressed as MDA
equivalents (nmol/ml) as described by Matsumoto
et al.
17
Lipoprotein(a) assay was carried out by immu-
noprecipitin analysis according to Gries et al.
18
with
reagents obtained ready for use from Incstar corp.
(Stillwater, MN, USA). Plasma levels of total choles-
terol,
19
high-density lipoprotein (HDL) cholesterol
20
and triglycerides
21
were estimated by kits of Bio-
Merieux Lab (France). Plasma LDL cholesterol was
calculated according to Friedewald et al.
22
Statistical methods
Statistical package of social sciences (SPSS) was
used for data management and analysis. Data were
Diagnostic value of nitric oxide, lipoprotein(a), and malondialdehyde levels
MA El-Latif et al
545
International Journal of Impotence Research
presented as mean7standard deviation (s.d.). The
independent sample t-test was used to compare the
results in two different groups. The paired t-test was
used to compare two variables in the same group.
Mann–Whitney U-test was used to compare non-
parametric data. The simple Pearson’s correlation
coefficient was used to evaluate the correlation
between two variables. Comparison of receiver
operating characteristic (ROC) area under curve
was performed by DeLong’s test. All tests were
considered statistically significant when Pwas
o0.05.
Results
Biochemical marker assays
Lipoprotein(a) levels were significantly higher in
the diabetic group (42.5716.5 mg/dl) vs the psycho-
genic (24.476.7 mg/dl) or control (23.577.01 md/
dl) groups (Po0.001 for diabetic vs other groups).
There was no difference in LP(a) between the
control or the psychogenic ED groups (P40.05).
Within the diabetic group, there was no difference
in LP(a) between men on insulin therapy, and those
on oral therapy only (41.0716.8 and 42.1717.1,
respectively) (Table 1).
Parallel changes were seen for venous MDA
levels. Diabetics had significantly higher venous
MDA (3.0170.45) compared to men in the psycho-
genic ED (1.0570.15) or control (1.0270.25) groups
(Po0.001 for diabetic vs any other group). In
contrast, peripheral NO levels were significantly
lower in the diabetic group (17.1277.69 mmol/l)
compared to the psychogenic ED (31.1377.4 mmol/
l) or control groups (28.678.7 mmol/l) (Po0.001 for
diabetics vs all other groups). There were no
differences between diabetics on insulin and those
not on insulin therapy in NO levels (15.777.7 vs
18.476.6, respectively, P40.05) nor in MDA levels
(2.9270.61 vs 2.9970.54, respectively, P40.05).
The source of the blood sample (peripheral vs
cavernous) did not affect the results of the marker
assays (Tables 1 and 2). The differences in periph-
eral marker levels observed between diabetic
and non-diabetic ED patients remained significant
when cavernous blood samples were analyzed
(Table 2).
Intracavernosal injection results
Intracavernosal injection results are summarized in
Table 2. There was a highly significant increase
in PSV in the psychogenic group compared to the
Table 1 Comparison of different biochemical markers levels in the three studied groups
Biochemical parameters (1) Diabetic (n¼26) (2) Psychogenic (n¼15) (3) Control (n¼10) P-value
1vs2 1vs3 2vs3
Venous lipoprotein(a) (mg/dl) 42.5716.5 24.476.7 23.577.01 o0.001 o0.001 NS
Venous NO (mmol/l) 17.1277.69 31.1377.4 28.678.27 o0.001 0.001 NS
Venous MDA (nmol/ml) 3.0170.45 1.0570.15 1.0270.25 o0.001 o0.001 NS
Hb.A1c (%) 10.9571.13 6.3470.19 6.1270.39 o0.001 o0.001 NS
Total cholesterol (mg/dl) 215.8751.48 160.8714.9 169.3712.86 0.001 0.008 NS
HDL cholesterol (mg/dl) 41.475.3 48.773.7 47.573.8 0.009 0.03 NS
LDL cholesterol (mg/dl) 145.7719.3 123.178.4 122.479.09 0.001 0.005 NS
Triglycerides (mg/dl) 129.9710.14 100.776.4 105.075.35 0.001 0.01 NS
Mean7s.d.
Abbreviations: HDL, high-density lipoprotein; LDL, low-density lipoprotein; MDA, malondialdehyde; NO Nitric oxide; NS,
nonsignificant.
Table 2 PPDU results and biochemical marker levels in venous and cavernous blood of diabetic vs psychogenic impotent patients
Parameters Diabetic group (n¼26) Psychogenic group (n¼15) P
PSV (cm/s) 16.674.7 31.376.3 o0.001
CAD increase (%) 53.0717.8 94.6718.6 o0.001
Venous lipoprotein(a) (mg/dl) 42.5716.5 24.476.7 o0.001
Cavernous lipoprotein(a) (mg/dl) 41.0716.0 24.1476.7 o0.001
Venous MDA (nmol/ml) 3.0170.45 1.0570.15 o0.001
Cavernous MDA (nmol/ml) 2.9570.58 1.1270.09 o0.001
Venous NO (mmol/l) 17.1177.69 28.1377.4 o0.001
Cavernous NO (mmol/l) 17.1577.17 27.877.44 o0.001
Mean7s.d.
Abbreviations: CAD, cavernosal artery diameter; NO, nitric oxide; PPDU, pharmaco-penile duplex ultrasonography; PSV, peak systolic
velocity.
Diagnostic value of nitric oxide, lipoprotein(a), and malondialdehyde levels
MA El-Latif et al
546
International Journal of Impotence Research
diabetic group (31.376.3 and 16.674.7 cm/s,
respectively, Po0.001, Table 2). Also, the percen-
tage of CAD increase after trimix injection was
higher in the psychogenic than in the diabetic group
(94.6718 and 53.0717.8%, respectively, Po0.001,
Table 2).
Correlation of biochemical markers and ICI results
within diabetic group
Within the diabetic group, there was a significant
positive correlation between venous LP(a) and the
duration of impotence (r¼0.59, Po0.05), and the
degree of glycemic control as measured by HgbA1c
(r¼0.69). The objective measures of PSV and
percent CAD increase after ICI also correlated
strongly with LP(a) levels (r¼0.81 and 0.88 for
PSV and %CAD, respectively, Po0.05).
There were significant correlations between ve-
nous NO levels and invasive testing results as well
(Table 3 and Figure 1b). While venous NO levels
correlated somewhat weakly with the duration of
impotence (r¼0.33), we found a strong positive
correlation of NO levels with subjective response to
ICI (r¼0.64, Po0.05), PSV (r¼0.75, Po0.05) and
percent CAD increase (r¼0.76, Po0.05).
These correlations were not affected by the source
of venous blood (peripheral vein vs flaccid penis)
(Table 3).
Biochemical markers as predictors of arterial
insufficiency
Using the definition of PSVo25 cm/s after ICI as
diagnostic of arterial insufficiency, 18 of 41 (44%)
impotent men were found to have arterial insuffi-
ciency. As a surrogate test, LP(a) had a 91%
sensitivity and a 78% specificity using a cutoff of
430 mg/dl to predict arterial disease, while MDA
was 91% sensitive and 89% specific at a cutoff of
1.7 nmol/ml. HbA1c was less accurate in predicting
low PSV with a sensitivity of 91% at HbA1c 47%,
but a specificity of only 44%. To compare the
various tests in a cutoff independent manner, ROC
area under curve were calculated (Table 4). These
show that LP(a) was the best test in predicting
Table 3 Correlation of both venous and cavernous NO and LP(a) with duration and severity of diabetes (a), and with results of ICI
testing (b)
Parameters Venous LP(a) Cavernous LP(a) Venous NO Cavernous NO
(a)
Duration of impotence (years) r¼0.59* r¼0.58* r¼0.33 r¼0.34
Hb.A1c% r¼0.69* r¼0.68* r¼0.53* r¼0.51*
(b)
PSV (cm/s) r¼0.81* r¼0.81* r¼0.75* r¼0.77*
CAD increase (%) r¼0.88* r¼0.89* r¼0.76* r¼0.78*
*¼Significant Po0.05.
Statistical significance was determined after applying the Holm correction for multiple testing.
Abbreviations: CAD, cavernosal artery diameter; LP(a), lipoprotein(a); PSV, peak systolic velocity.
0
1
2
3
4
5
6
Peak systolic velocity (cm/sec)
Malondialdehyde
(nmol/ml)
0
10
20
30
40
50
60
70
0 1020304050
0 1020304050
0 1020304050
0 1020304050
Peak systolic velocity (cm/sec)
Lipotrein (a)
(mg/dl)
0
5
10
15
20
Peak systolic velocity (cm/sec)
HbA1c (%)
0
10
20
30
40
50
Peak Systolic Velocity (cm/sec)
Nitric Oxide
(micromol/ml)
Figure 1 Scatter-plots of biochemical markers in all 41 impotent patients vs peak systolic velocity obtained with invasive testing.
Diagnostic value of nitric oxide, lipoprotein(a), and malondialdehyde levels
MA El-Latif et al
547
International Journal of Impotence Research
arterial insufficiency, and was superior to HbA1c
(P¼0.006).
Discussion
Erectile dysfunction is a common pathological
development in individuals with DM.
1,2
It is
thought to be mainly due to a combination of
vascular and neurogenic changes
3
and is strongly
associated with other risk factors of cardiovascular
disease.
Lipoprotein(a) levels have been suggested to be an
independent risk factor for atherosclerosis,
10
which
is, in turn, highly associated with ED. In our study,
we found significantly higher levels of LP(a) and
total cholesterol in the diabetic group than in both
the psychogenic impotent and the healthy control
groups, while the difference between the latter two
groups was non-significant. The increased levels
of LP(a) correlated with poor response to ICI as
measured by CAD increase and PSV, in agreement
with others.
23
These findings also confirm previous
studies suggesting that ED in diabetic patients is
associated with impaired penile arterial flow.
7,24
This can be explained by atherosclerosis of large
arteries and microangiopathy of small arteries
occurring in DM,
4
and we propose that increased
LP(a) may be a marker of these changes.
Development and maintenance of penile erection
requires the relaxation of the smooth muscle cells in
the cavernous bodies and is essentially mediated by
NO.
7,25
During sexual stimulation, NO is synthe-
sized from L-arginine by nitric oxide synthases of
neuronal (nNOS) and endothelial (eNOS) ori-
gins.
25,26
In this study, we found a significant
decrease of cavernosal and peripheral NO in
diabetic patients, but not in patients with psycho-
genic ED, in agreement with others.
27
This finding
could be due to the presence of advanced glycosyla-
tion end-products (AGEs), compounds that are
formed as a result of non-enzymatic reaction
between glucose and the amino groups of proteins.
Advanced glycosylation end-products have been
shown to quench NO and result in attenuation
of NO-mediated relaxation of cavernosal smooth
muscle.
28,29
More recently, it was shown that eNOS
itself is modified by addition of O-GlcNAc residue
in a rat model of diabetic ED.
25
As a result,
endothelial production of NO in response to shear
stress, believed to be necessary for maintaining
erections, was impaired.
25
Another possible mecha-
nism of reduced NO in diabetics is competition
for the Arginine substrate be Arginase II, which
has been shown to be upregulated in the corpus
cavernosum of diabetics.
8,30
Increased oxidative stress via the production of
ROS has been reported in the diabetic state.
13,30
Our
studies confirm that increased oxidative stress, as
measured by MDA levels, correlates with poor
erectile response in human subjects. In addition,
our study agrees with Sozmen et al.,
31
who reported
that poor glycemic control is strongly associated
with an increase in free radicals and diabetic
complications, and extends their findings to ED.
Others have studied the mechanisms by which
oxidative stress could cause ED.
12
In a rabbit model
of diabetic ED, Khan et al.
32
found that increased
levels of superoxide anion in diabetes can divert
NO away from the erectogenic pathway through
its conversion to peroxynitrite, and that addition
of superoxide dismutase enhanced NO-mediated
muscle relaxation.
31
Similarly, supplementation
with vitamin E, a free radical scavenger, was found
to increase PDE5 inhibitor-mediated erection in
diabetic rats.
33
The results of our study showed, also, a non-
significant difference between both peripheral
venous and cavernous NO in diabetic patients. The
high correlation between cavernosal and venous
values of the biochemical markers suggest that these
changes are reflective of systemic changes, and are
not selective for the cavernosal tissues.
5
In addition,
the present studies do not establish that these
markers are selective for erectile dysfunction among
the general diabetic population, since we did not
include potent diabetic patients as controls. This
was a reasonable approach given the invasive
nature of penile arterial testing and cavernosal
blood sampling. Still, the markers studied were
specific to arterial dysfunction among diabetic men,
to a degree not predicted by glycated hemoglobin
alone.
So, we can conclude that the finding of high levels
of LP(a) and MDA with low levels of NO in
peripheral venous blood correlates with the severity
of ED as measured by PPDU testing. However, we
should caution that these assays are not currently
standardized. Therefore, it may be more appropriate
to use them as an adjunct, rather than a replacement,
to invasive testing at this time. An additional
limitation is that we have chosen to apply extensive
exclusion criteria, especially smoking and overt
cardiovascular disease, in order to avoid potential
Table 4 ROC area under curve for biochemical markers vs
invasive testing results in all impotent men (n¼41)
Marker ROC area Pvs HbA1c
Cholesterol 0.71 0.34
Triglycerides 0.68 0.13
HbA1c 0.74 —
LP(a) 0.95 0.006
MDA 0.92 0.01
NO 0.92 0.01
Pcalculated by DeLong’s test.
Abbreviations: LP(a), lipoprotein(a); MDA, malondialdehyde;
NO, nitric oxide; ROC, receiver operating characteristic.
Diagnostic value of nitric oxide, lipoprotein(a), and malondialdehyde levels
MA El-Latif et al
548
International Journal of Impotence Research
confounders. One drawback of this approach, how-
ever, is the limitation of the ability to generalize the
present findings to the entire population of diabetic
men with ED. Our findings provide a rationale for
future studies of biochemical markers in a wider
population of diabetic men with ED.
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