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(Hypertension. 1997;30:405.)
© 1997 American Heart Association, Inc.
Articles |
Reduced Plasma Concentrations of Nitrogen Oxide in Individuals With Essential Hypertension
From the First Department of Medicine (K.N., M.K., M.H.) and the Department of Physiology (H.K.), Osaka University School of Medicine; and the Sumitomo Life Multiphasic Health Test System (H.Y.), Osaka, Japan.
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Abstract |
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Abstract Patients with essential hypertension exhibit blunted endothelium-dependent vasodilator responses, which may be largely attributable to reduced bioactivity of nitric oxide (NO). Therefore, we measured the end product of NO, nitrate plus nitrite (nitrogen oxide), and examined the relationship between the degree of hypertension and plasma nitrate plus nitrite levels in patients with essential hypertension. The combined plasma concentration of nitrate plus nitrite, end products of NO metabolism, was reduced in individuals with essential hypertension relative to that in control subjects (15.7±1.1 versus 22.8±1.4 mmol · L-1, P<.001); individuals with borderline hypertension showed values that were intermediate between those of the other two groups (18.2±1.2 mmol · L-1, P<.001). The plasma nitrogen oxide concentration showed significant inverse correlations with both systolic and diastolic blood pressures. The basal concentration of nitrogen oxide in the plasma was reduced, at least in the peripheral circulation, in individuals with essential hypertension.
Key Words: hypertension, essential • risk factors • blood pressure • endothelium-derived factors
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The vascular endothelium plays a central role in the regulation of regional blood flow by the release of endothelium-dependent relaxing factor,1 of which nitric oxide (NO) is a major component.2 The critical role of endothelium in the regulation of vascular tone is emphasized by the association of abnormal endothelial function with cardiovascular disease. Essential hypertension is characterized by an increase in vascular tone that results in an increase in vascular resistance. In spontaneously hypertensive rats, basal production of NO is reduced.3 In patients with essential hypertension, endothelium-dependent vasodilator responses are attenuated,4 5 which is largely attributable to reduced bioactivity of NO.6 Falloon and Heagerty7 also provided evidence for defective endothelium-dependent dilatation in resistance arteries of patients with essential hypertension. However, Cockcroft et al8 used a standard forearm vascular resistance approach and found no difference in the vasodilator responses to acetylcholine or carbachol between patients with essential hyper- tension and matched normotensive control subjects. Because the studies of Falloon and Heagerty7 and Cockcroft et al8 used indirect assessment of the involvement of NO in essential hypertension, it may be preferable to measure NO directly to determine the involvement of NO. NO released from cells rapidly autoxidizes to yield nitrite (NO2-), which interacts with hemoglobin to yield nitrate (NO3-). Because nitrite plus nitrate (nitrogen oxide) is relatively stable in blood, the level of nitrate plus nitrite in blood may be an indicator of the endogenous formation of NO. Therefore, we measured the end product of NO, nitrate plus nitrite, and examined the relationship between the degree of hypertension and plasma nitrate plus nitrite levels in patients with essential hypertension.
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Methods |
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Subjects and Study Design
This study was approved by our institutional committee on clinical investigation. All subjects gave their written informed consent for participation. Normal blood pressure was defined as systolic pressure <140 mm Hg and diastolic pressure <90 mm Hg. Hypertension was defined as either systolic pressure
160 mm Hg or diastolic
pressure of 95 mm Hg, or both, with a well-documented history
of long-term high blood pressure. The hypertension group comprised 108
patients (78 men and 30 women with a mean±SEM age of 49±3 years).
Borderline hypertension was defined as blood pressure between the
normal and hypertensive ranges. The 91 patients (67 men and 24 women)
in this category were aged 48±2 years; causes of secondary
hypertension, such as pheochromocytoma, renovascular disease,
hyperthyroidism, and aortic coarctation, had been excluded in all
patients by the primary physician on the basis of conventional clinical
and laboratory criteria before the initiation of antihypertensive
therapy.
A total of 127 normal subjects (81 men and 46 women) aged 50±3 years
were matched with the patients for sex and approximate age and served
as the control group. We selected patients who had received no previous
treatment with antihypertensive drugs and the other few patients whose
antihypertensive drug treatment had been washed out for at least 2
weeks. Clinical history, physical examination,
electrocardiography, chest
radiography, and routine laboratory tests revealed no
evidence of present or past diabetes mellitus;
hypercholesterolemia;
cardiovascular diseases; a body weight of >120% of
the normal value; smoking; abnormality in liver, renal, or thyroid
function; or any disease conferring a predisposition to vasculitis or
Raynaud’s phenomenon. No patient showed ST-segment elevation during an
upright bicycle exercise test. Participants were instructed to refrain
from eating for 18 hours, drinking beverages containing alcohol or
caffeine, or smoking for at least 24 hours before blood sampling. In a
preliminary study, we measured plasma nitrogen oxide levels in young
healthy volunteers at 1, 3, 6, 12, and 18 hours after they had been
eating a regular Japanese diet (n=7) and confirmed that at 1 hour after
eating, the plasma nitrogen oxide levels increased from 
24
mmol · L-1 to 33 mmol ·
L-1 (P<.05); after 12 and 18
hours, they decreased to the baseline levels. Therefore, the
contribution of nitrate in the diet to plasma nitrate concentration was
minimized by asking subjects not to eat foods for 18 hours or not to
drink for 6 hours.
NO Measurement
Specimens (1.5 mL) of peripheral venous blood from
the brachial vein were collected into heparinized tubes after the
subjects had been sitting at rest for 15 minutes in a quiet room
maintained at a temperature of 22°C to 24°C. The blood was placed
immediately in an ice bath and centrifuged within 30 seconds
for 5 minutes at 2000g. The serum fraction was diluted 1:1
with nitrite- and nitrate-free distilled water, and 400 mL of the
diluted sample was centrifuged at 2000g in an
ultra-free MC microcentrifuge device (Millipore) to remove
substances larger than 10 kD. The filtrate was passed through a
copper-plated cadmium column to reduce nitrate to nitrite and then
reacted with Griess reagents consisting of 0.1%
naphthylethylenediamine dihydrochloride in distilled water and 1%
sulfanilamide in 5% H3PO4, after which
absorbance was measured at 540 nm9 to provide the total
amount of plasma NO end products (nitrate plus nitrite). The
efficiency of the cadmium column in the conversion of nitrate to
nitrite was confirmed to be 100% by measuring both nitrate and nitrite
standards before and after sample measurement.9
Statistical Analysis
Data are expressed as mean±SEM. Differences in plasma nitrogen
oxide concentrations among groups were assessed by ANOVA followed by
Bonferroni’s test. A value of P<.05 was considered
statistically significant.
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Results |
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The clinical characteristics of hypertensive patients and normal control subjects are summarized in the Table. With the exception of systemic blood pressure (measured at the time of the study), no significant differences in these characteristics were observed between the patient and control groups. There were no significant differences in plasma nitrogen oxide among the various drug-treated groups and the not-treated group. However, the concentration of nitrogen oxide (nitrate plus nitrite) in the plasma of systemic venous blood was significantly more reduced in the hypertension group than in the control group (Fig 1). The plasma concentration of nitrogen oxide in the borderline hypertension group was intermediate between the values for the control and hypertension groups. The plasma nitrogen oxide concentration showed a significant inverse correlation with both systolic and diastolic blood pressures (Fig 2) but was not correlated with the plasma concentration of creatinine (r=.12).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The plasma concentration of nitrogen oxide in systemic venous blood is determined by synthesis, degradation, and clearance of NO. In the present study, renal function as assessed by creatinine clearance among the three groups and plasma nitrogen oxide concentration did not correlate with the creatinine, suggesting that the decrease in serum nitrate plus nitrite concentration in hypertensive patients resulted predominantly from a reduced synthesis or an increased degradation of NO. Daily activity and the consumption of food or water may also affect nitrogen oxide concentration. However, we confirmed that there are no aftereffects of eating and drinking following 8 hours of fasting or 6 hours of abstaining from drinking, as is shown in "Methods." As for the synthesis of NO, NO is continuously synthesized from L-arginine in a reaction catalyzed by NO synthase, with most NO present in the circulation originating from endothelial and smooth muscle cells. Hypertension can produce structural damage to aortic endothelial cells in animals, and pressure overload is associated with a direct toxic effect on human endothelium; impairment of the release of NO from vascular endothelial cells may thus contribute to the reduced plasma nitrogen oxide concentrations in patients with essential hypertension. Decreased synthesis of NO might also result from abnormal handling of intracellular calcium and a consequent reduction in the activity of NO synthase.10 However, this study failed to detect a link between the endothelial NO synthase gene to essential hypertension.11 Hypertension impairs endothelium-dependent dilation of rat coronary arteries as a result of superoxide anion–mediated degradation of NO.12 Indeed, increased production of superoxide anions, which rapidly deactivate NO, is a characteristic feature of experimental models of hypertension,12 13 and plasma indexes of lipid peroxidation are increased in patients with hypertension.14 However, we could not clarify the major factor for the reduction of plasma nitrogen oxide, and it remains unknown whether the decrease in plasma nitrogen oxide levels was the cause or effect of impairment of endothelial function. The decreased plasma nitrogen oxide levels may not even reflect the impairment of endothelial function because NO is produced not only at endothelial cells but also at leukocytes, platelets, nerves, cardiomyocytes, and muscles.
The plasma concentrations of various vasoconstrictors, including norepinephrine, angiotensin, and endothelin,15 are increased in individuals with hypertension. On the other hand, shear stress in the blood vessels16 is also increased, which may stimulate NO production. Xiao and Pang17 showed that NO synthesis in vascular smooth muscle cells increases during development in spontaneously hypertensive rats. Although the basal release of NO is not impaired, NO may be functionally ineffective with regard to its hemodynamic role in the systemic vasculature in spontaneous hypertensive rats.18 These apparent discrepancies may be attributable to the differences in species, in genetic versus essential hypertension, and in the extent or duration of hypertension or vascular wall injury.
Among the hypertensive subjects, there remains the difference in the duration of the hypertensive state, and daily physical activity or menstrual cycle19 may have influenced the nitrogen oxide levels. These may be limiting factors in the determination of the accurate value of nitrate levels in the present study, and future well-controlled studies need to be conducted.
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Footnotes |
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Reprint requests to Masafumi Kitakaze, MD, First Department of Medicine, Osaka University School of Medicine, 2-2 Yamadaoka, Suita 565, Japan.
Received November 11, 1996; first decision December 2, 1996; accepted February 18, 1997.
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