(Circulation. 1995;91:2982-2988.)
© 1995 American Heart Association, Inc.
Articles |
Pharmacodynamics of Plasma Nitrate/Nitrite as an Indication of Nitric Oxide Formation in Conscious Dogs
From the Department of Physiology, New York Medical College, Valhalla.
Correspondence to Thomas H. Hintze, PhD, Professor, Department of Physiology, New York Medical College, Valhalla, NY 10595.
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Abstract |
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Background The present investigation was undertaken to better understand the production of nitric oxide (NO) in vivo as measured by alterations in plasma nitrite or nitrate in blood samples from studies in experimental animals or clinical studies in humans.
Methods and Results Plasma samples were taken from the aorta, the coronary sinus, a peripheral vein in the leg (skeletal muscle), or the right ventricle (mixed venous) in chronically instrumented conscious dogs. Plasma nitrite was converted to NO gas in an argon environment by use of hydrochloric acid, and plasma nitrate was converted first to nitrite with nitrate reductase and then to NO gas with acid. Standard curves were constructed, and the amount of nitrite and nitrate in plasma was determined. The primary metabolite was nitrate, whereas nitrite was approximately 10% of the total and remained constant. In the resting dog, the only vascular bed with a positive arterial-venous nitrate difference, evidence for production of NO, was the heart. Nitrate infusion into quietly resting dogs resulted in increases in plasma nitrate up to 38±3.4 mmol/L, increases in systemic arterial pressure, and a marked diuresis. The plasma half-life was calculated as 3.8 hours. The volume of distribution was calculated as 0.215 L/kg, or equivalent to the extracellular volume.
Conclusions These studies indicate that nitrate is a reliable measure of NO metabolism in vivo but that because of the long half-life, nitrate will accumulate in plasma once it is produced. Because of the large volume of distribution (21% of body weight versus the 4% of body weight usually attributed to plasma volume, the compartment in which nitrate is measured), simple measures of plasma nitrate underestimate by a factor of 4 to 6 the actual production of nitrate or NO by the body. In disease states, such as heart failure, in which renal function and extracellular volume are altered, caution should be exercised when increases in nitrate in plasma as an index of NO formation are evaluated.
Key Words: endothelium-derived factors • diuretics • hypertension
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Introduction |
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Since the discovery of endothelium-derived relaxing factor (EDRF) by Furchgott and Zawadski1 and its identification as nitric oxide (NO) as reviewed by Vanhoutte,2 Ignarro et al,3 Moncada,4 and many others, increasing evidence suggests that NO is essential for normal cardiovascular control. For instance, blockade of EDRF synthesis results in a rapid and marked increase in arterial pressure and vascular resistance.5 6 In addition, it is probable that an increased or reduced production of NO contributes at least in part to a number of pathophysiological states. NO production may be elevated, leading to the hypotension of septic shock7 ; NO production may be low, as we have recently reported,8 in heart failure, leading to reduced coronary vasodilation to acetylcholine; and NO production may be induced with aerobic exercise9 10 and be responsible for the beneficial cardiovascular effects of exercise training.
A number of potential active intermediates from NO may have potent biological activity.11 NO appears in biological fluids as dissolved NO gas, in aqueous solution as nitrite anion, and in blood samples or plasma as nitrate anion12 ; measurement of these may reflect changes in NO production by tissues. Recent studies from a number of laboratories have presented data from humans indicating that the chief metabolite of NO in plasma, NO3- or nitrate anion, is elevated, low, or normal.13 14 15 Some assumptions inherent in the measurement of a metabolic product in plasma or blood as an index of an altered metabolic state are that (1) the plasma half-life is long or at least known; (2) the volume of distribution is known; (3) the measurement of a single NO metabolic product reflects the concentration of the substance in question; (4) all of the sources of the metabolite are known and accounted for; and (5) the biological activity of the metabolite may contribute to the observed physiology of the substance. With these considerations in mind and with the objective of developing a reliable method to assess changes in NO production in vivo, the goal of the present study was to understand the production, pharmacodynamics, and biological activity of nitrite and nitrate anions in conscious dogs.
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Methods |
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Measurement of Plasma Nitrite/Nitrate
Briefly, plasma nitrite was measured by acidifying plasma to pH <2.0 to convert nitrite to NO. Plasma nitrate was measured by incubating plasma with Aspergillus nitrate reductase (Boehringer Mannheim) to reduce nitrate into nitrite and then converting nitrite into NO by the addition of hydrochloric acid. The NO produced was then injected into the NO analyzer (Sievers, Inc), and the NO content of the sample was determined by measuring the luminescence generated in the presence of ozone. The luminescence measured was directly proportional to the amount of NO injected and, in turn, to the nitrite or nitrate content of the sample.
Blood was obtained from conscious dogs in heparinized tubes for plasma standards or samples. Heparin was used as an anticoagulant instead of EDTA because the nitrate reductase used to convert nitrate to nitrite is a calcium-dependent enzyme. The blood was centrifuged at 1000g for 15 minutes to remove formed elements, and the plasma was frozen for at least 24 hours before analysis. On the day of analysis, plasma samples were thawed and vigorously vortexed for 10 seconds. The precipitated proteins were removed by centrifugation (1000g for 30 minutes), and 1 mL of supernatant was placed into a 5-mL polypropylene tube. Each sample was divided into three aliquots: one to measure NO, one to measure plasma nitrite, and one to measure plasma nitrate plus nitrite. The concentration of nitrate was then calculated by subtracting the amount of nitrite from the value of the tube containing both nitrite and nitrate. All samples were run in triplicate.
For measurement of plasma nitric oxide, plasma samples were incubated under argon, and headspace gas was removed to measure plasma NO.
For analysis of nitrite, 1 mL of standard or sample was placed in a 5-mL tube, capped, and degassed as described above. After 15 minutes of degassing, 40 µL HCl was added, and the samples were incubated for 5 minutes at 36°C. The content of NO was quantified.
For measurement of plasma nitrate plus nitrite, nitrate was reduced to nitrite by adding to each tube 10 µL (0.2 U) Aspergillus nitrate reductase (reconstituted in distilled water as 20 U/mL), 10 µL (0.1 µmol) FAD, and 10 µL (0.1 µmol) reduced NADP (Boehringer Mannheim Biochemicals). Tubes were capped immediately with a rubber septum (Suba-Seal Septa, Aldrich Chemical Co), and the oxygen in the headspace above the sample was removed and replaced with argon. Samples were incubated at 36°C for 1 hour under the inert argon atmosphere to allow conversion of nitrate to nitrite. After incubation, 40 µL concentrated HCl was added through the seal to each reaction tube to achieve a pH <2. Tubes were incubated for an additional 5 minutes at 36°C. A 500-µL sample of the headspace gas was removed from the sealed tubes at the end of the incubation and injected into the NO analyzer to quantify the NO released into the headspace gas.
Nitrate and Nitrite Standard Curves
Standard curves for
nitrite (0 to 10 µmol/L) and nitrate (0 to
80 µmol/L) were constructed by adding small aliquots (10-µL/mL
sample) of potassium nitrite or sodium nitrate solution (in distilled
water) to 1 mL pooled plasma. All standards were analyzed as described
above for plasma samples. A standard curve relating the luminescence
produced by the added nitrite or nitrate (Fig 1
) was
constructed, and the data were fit to a straight line (linear
regression). Only standard curves where r>.90 were used.
The slope relating the change in luminescence above basal levels to the
concentration of nitrite or nitrate was used to determine the nitrite
and nitrate content in plasma samples. To calculate the interassay
coefficient of variation, 50 1-mL plasma aliquots were collected from a
single dog, and 3 of these were run for the measurement of nitrite and
nitrate in every assay performed. To calculate the intra-assay
coefficient of variation, the basal nitrate and nitrite in each assay
was run in triplicate, and the differences were compared.
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Preparation of Dogs
Male mongrel dogs were instrumented after
sedation with
acepromazine maleate (1 mg/kg IV) followed by general anesthesia with
pentobarbital sodium (25 mg/kg IV). A left lateral thoracotomy was
performed, and indwelling catheters were placed into the ascending
aorta (22 dogs), coronary sinus (7 dogs), or right ventricle (7 dogs).
Catheters were also placed in the femoral or radial vein (7 dogs). The
chest was closed in layers, and catheters were exteriorized. The
animals were allowed to recover for 2 to 3 weeks, during which time the
animals were trained to lie quietly on the laboratory table. The
protocols were approved by the Institutional Animal Care and Use
Committee of New York Medical College and conform to the Guiding
Principles for the Use and Care of Laboratory Animals of the American
Physiological Society and the National Institutes of Health.
On the day of the experiment, the animals were brought to the laboratory and allowed to lie quietly on the laboratory table. Heart rate and blood pressure were monitored continuously. In some of the studies, female dogs were used, and the urinary bladder was cannulated for 1 day with a Foley catheter (12F). We have used all these techniques previously.5 8 9 10 16
Determination of Basal Nitrate and Nitrite Content in Plasma
Blood samples were obtained from the thoracic aorta, coronary
sinus, right ventricle, and femoral or radial vein. The plasma was
separated and kept frozen at -20°C until the time of assay.
Determination of Plasma Nitrate During Intravenous Infusions of
Nitrate
Sodium nitrate was dissolved in sterile normal saline (in
appropriate concentrations as mg · mL-1 · kg
body
wt-1) and infused into the femoral vein (1.0 mL/min) of
conscious dogs at a rate equivalent to 0.05, 0.10, 0.50, and 1.0
mg · kg-1 · min-1 for 10
minutes at
each step with a calibrated infusion pump (Harvard). Heart rate and
mean arterial pressure were recorded. During each steady state and at
the end of the infusion period, blood samples were taken from the aorta
and femoral vein. All plasma samples were processed for measurement of
nitrate.
Determination of Nitrate Plasma Half-life
Nitrate was infused
at a rate of 50
mg · kg-1 · min-1 for 15
minutes at a
rate of 2.0 mL/min to reach a steady state. Blood pressure and heart
rate were continuously recorded. Plasma samples were obtained at 0, 1,
3, 5, 10, 30, 60, 90, 120, 150, 180 (n=8), and 360 and 1440 (24 hours,
n=4) minutes after infusion. In the female dogs, the bladder was
catheterized, and urine was collected before the infusion (2x20
minutes) at 5-minute intervals for 1 hour and at 15-minute intervals
for an additional 90 minutes and during the last 30 minutes (a total of
3 hours). The amount of urine collected was divided by the time
interval to estimate the average urine flow rate. We have used these
techniques previously to determine the half-life of atrial natriuretic
factor.17
For the determination of nitrate content in these samples, it was necessary to dilute the plasma samples. Plasma samples were diluted 100-fold with distilled water. A separate standard curve was constructed in a similar manner by preparation of a concentrated plasma sample and then serial dilution with untreated plasma to obtain concentrations over the range of 25 to 400 µmol/L. In addition, the sample and standards were incubated in the presence of 0.2 µmol NADPH and 500 µL of the diluted plasma. A 200-µL aliquot of headspace gas was injected into the NO analyzer. The content of NO in the treated samples was determined from the standard curve prepared in a similar fashion.
Calculation of Plasma Half-life
The plasma concentration
obtained at each time period was
determined, and the average values were plotted and used to estimate
the time constant, assuming a single exponential decay of the plasma
content with time. We have used these techniques
previously.17
Calculation of Volume of Distribution of Nitrate
The initial
plasma concentration at time 0 was estimated from
the exponential equation of the plasma half-life using the parameters
from the fitted equation. The amount infused divided by the plasma
concentration at time 0 results in the volume of distribution. This was
compared with the total body water or plasma volume to estimate the
compartment in which the nitrate was distributed. The total body water
and plasma volume were estimated as 65% and 4%, respectively, of the
body weight of the dogs.
Statistical Analysis
All data are expressed as the
mean±SEM. To estimate the amount
of nitrate or nitrite produced by an organ, the arterial or venous
plasma concentration was multiplied by the measured blood flow or blood
flow previously determined in conscious dogs in our
laboratory.8 9 10 For instance, arterial
and coronary sinus
concentrations were multiplied by 37 mL/min (left circumflex coronary
blood flow), arterial and right ventricular concentrations were
multiplied by 2500 mL/min (cardiac output), and arterial and hindlimb
venous samples were multiplied by 600 mL/min (terminal aortic blood
flow). This results in estimates of coronary, systemic or pulmonary,
and skeletal muscle NO production, respectively. Comparisons between
groups of samples were performed by paired Student's t test
or repeated-measures ANOVA, as appropriate. Specific comparisons
between means were done with Student-Newman-Keuls multiple-range tests.
A value of P<.05 was considered statistically significant.
The time constant for determining the plasma half-life of nitrate, the
half-time for arterial pressure, and urine flow rate were determined by
nonlinear curve fitting of the averaged data to a single exponential
equation using the Levenberg-Marquardt algorithm to determine the
coefficients of the equation (SLIDEWRITE PLUS 2.0 for
Windows, Advanced Graphics Software, Inc). The slope, intercept, and
r value for nitrite and nitrate or the relation between
arterial pressure and plasma nitrate were calculated by a linear
regression analysis, and all illustrations were produced with
SlideWrite Plus 2.0 for Windows. The coefficients of variation were
calculated as the SD/meanx100.
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Results |
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Nitrate and Nitrite Standard Curves
The relations between nitrate or nitrite and chemiluminescence are shown in Fig 1A
and 1B, respectively. There were
good correlations
between the concentration of nitrate or nitrite added to plasma and the
luminescence over the expected plasma range of nitrite or nitrate.
There was essentially no luminescence by plasma incubated by itself,
indicating that the amount of free NO in plasma is below the detection
limit of our method. The plasma concentrations of nitrite (n=8) for
calculation of intra-assay and interassay coefficients of variation
were 1.06±0.09 and 1.13±0.12 µmol/L, and the coefficients of
variation were 24% and 35%, respectively. The plasma concentrations
of nitrate (n=8) were 9.73±0.87 and 10.8±1.31 µmol/L,
and the
coefficients of variation for intra-assay and interassay variations
were 25% and 38%, respectively.
Determination of Basal Nitrate and Nitrite Content in Plasma
Because we collected the greatest number of samples from the aorta
(n=22), we constructed the frequency relation between measured nitrite
or nitrate in each dog and the number of dogs having that plasma level.
The results are shown in Fig 2. Plasma nitrite was
always lower than plasma nitrate in our conscious dogs: The mean plasma
nitrite was 1.0±0.2 µmol/L and the mean plasma nitrate
9.0±1.0
µmol/L. Similar results, although with much smaller sample sizes
(generally n=7), were found in mixed-venous or coronary sinus plasma
samples (data not shown).
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We estimated the amount of nitrate produced
by the pulmonary
circulation, the coronary circulation, and the skeletal muscle
circulation by taking arterial and venous samples across these vascular
beds and multiplying by the blood flow (from the literature). The blood
flow times the arterial concentration is the delivery (input), and the
blood flow times the venous concentration is the amount added or
subtracted from the arterial blood (output) in moles per minute. The
data are shown in Fig 3. There was no significant
difference in the input or output of nitrate in the pulmonary
circulation or the skeletal muscle circulation, but there was a
statistically significant increase in the coronary circulation. Similar
results were found for the sum of nitrite and nitrate or for nitrite
alone, although much lower, since the concentration of nitrite in
plasma is approximately 10% of the nitrate concentration. There was no
significant correlation between the amount of nitrite measured and the
amount of nitrate measured in plasma samples. This is because the
relation, as shown in Fig 4, is a flat line, indicating
that nitrite is constant over a large range of plasma nitrate
concentrations. Exponential and logarithmic plots were also attempted,
and there was also no significant fit of the data. Thus, the
measurement of nitrite by itself is not necessary, and the one tube
containing both nitrate and nitrite (addition of nitrate reductase plus
acid) is sufficient to measure all the changes in plasma NO.
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To determine whether sampling of venous plasma was indicative of NO production by an organ, we determined the relation between venous plasma nitrite plus nitrate and the arterial-venous difference across organs. There was no correlation (y=0.2x-0.99, r=.2, P=.61) between peripheral venous nitrite plus nitrate and the systemic arterial-venous difference, whereas there were good correlations between pulmonary and mixed-venous–arterial (pulmonary circulation, y=0.94x-7.3, r=.77, P<.001) and coronary sinus and arterial-coronary sinus (coronary circulation, y=0.74x-4.97, r=.92, P<.001).
Determination of Plasma Nitrate During Intravenous Infusions of
Nitrate
The relation between plasma nitrate and the infusion rate of
nitrate is shown in Fig 5. There were significant
increases in arterial, aortic, and peripheral venous (skeletal muscle
vein) nitrate during infusion. There were also significant increases in
systemic arterial pressure and reductions in heart rate during the
infusion of nitrate. There was a significant linear correlation between
plasma nitrate and arterial pressure in our conscious dogs
(y=1.51x+108, r=.99,
P<.001).
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Calculation of Plasma Half-life
In the dogs used to calculate
the plasma half-life of nitrate, the
infusion of nitrate at 50
mg · kg-1 · min-1 for 15
minutes
resulted in an increase of plasma nitrate from 0.012±.0012 to
38±3.4
mmol/L. This was accompanied by an increase in mean arterial pressure
from 106±2 to 166±6 mm Hg (P<.05). There was also a
marked increase in urine flow rate, as shown in Fig 6.
Once the infusion of nitrate was stopped, there was a relatively rapid
return of mean arterial pressure (Fig 7), with a
half-time of 29.6 minutes (n=8). The diuresis continued for more than 3
hours (Fig 6) and had a calculated half-time of 74.6 minutes.
The fall
in plasma nitrate with time is shown in Fig 8. There was
a good monoexponential fit for the relation between plasma level and
time (r=.986). The estimated half-life was 230 minutes, or
3.8 hours.
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Calculation of Volume of Distribution of Nitrate
Using the
monoexponential equation from Fig 8, we calculated the
volume of distribution of nitrate in the conscious dog. The
concentration at time zero was 38±3.4 mmol/L, and the total amount
infused was 0.203 mol. Thus, the volume of distribution is 0.215 L/kg,
or 21.5% of the body weight. This is recognized as a reasonable
estimate of extracellular water and is larger than plasma volume (the
compartment in which nitrate is usually measured, 4% of body weight)
but less than total body water (65% of body weight).
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Discussion |
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A number of important conclusions in our study reflect not only the reliability of nitrate as a measure of NO production in vivo but also our understanding of the quantitative production of NO as reported in both animal and clinical studies. First, nitrate is the primary metabolite, and the concentration of nitrite in plasma is essentially constant. Second, the half-life of nitrate is long, 3.8 hours, indicating that after stimulation of NO formation, nitrate will remain in plasma. This may lead to an actual accumulation of nitrate anion if repeated stimuli for NO formation are used within a short period of time. Third, because of the large volume of distribution of nitrate, ie, the extracellular space, small steady-state changes in plasma or blood nitrate do not accurately reflect the actual production rate of NO (moles per minute), since the plasma volume is a small fraction of the extracellular volume. Fourth, a single measure of venous or arterial plasma nitrate, without knowledge of the arterial-venous concentration difference over time, may not reflect an increased production of NO, since nitrate may just be accumulating with time and not be constantly produced. Finally, high levels of plasma nitrate result in an increase in arterial pressure and diuresis. This supports previous studies, primarily from the 1950s, designed to study the toxicity of nitrate in humans.18 19 Furthermore, infusions of sodium nitrate are known historically as diuretics that cause a marked chloruresis, much like furosemide, since nitrate is reabsorbed preferentially over chloride anion in the kidney.18
The procedure we have developed to measure nitrite and nitrate is an adaptation of previously published methods.20 21 22 Incubation of plasma under an argon atmosphere by itself, in the presence of acid, and in the presence of nitrate reductase followed by addition of acid resulted in the production of NO gas that diffused into the headspace gas phase. Argon was used to prevent the reoxidation of NO gas to reform nitrogen dioxide. A sample of headspace gas was then injected with a gas-tight syringe into the NO analyzer, which resulted in the generation of sharp peaks that were easily integrated. Plasma was spiked with known amounts of nitrite or nitrate, and the samples were treated as unknowns. This resulted in good external standard curves; that is, linear relations were calculated between luminescence and NO2-/NO3- concentration over the range of 0.5 to 120 µmol/L. Since all the incubations are performed in capped tubes under an argon atmosphere, a large number of tubes, ie, 40 to 50, can be prepared and the incubations performed simultaneously. In addition, since a fixed volume of the headspace gas, containing NO from the sample in an argon atmosphere, was injected rapidly into the NO analyzer, there was a symmetrical peak that lasted for 1 to 2 minutes. This allowed for a large number of samples to be studied (approximately one every 3 to 4 minutes) and also allowed for varying the amount of gas injected so that the size of the peak could be optimized for each sample. This is a distinct advantage compared with other methods,23 24 such as a cadmium reflux column, in which the evolution of NO gas in the reflux chamber is slow, sometimes resulting in an asymmetrical peak that is difficult to integrate accurately and in potential contamination of the catalyst and/or detector with biological fluids.25
In plasma samples from the aorta, coronary sinus, peripheral vein, or
right ventricle, the primary metabolite of NO metabolism was nitrate,
with <10% of the total appearing as nitrite and no detectable NO.
This has been reported previously by a number of
laboratories.11 13 14 15 22
Furthermore, the amount of
nitrite was constant over a plasma range of nitrate from 5 to 
20
µmol/L. By multiplying the amount of nitrate or nitrite by blood flow
values previously recorded in conscious dogs from our laboratory or the
literature, we calculated the number of moles of nitrate, nitrite, or
both entering a specific vascular bed and the amount leaving. The only
vascular bed in which there was a statistically significant increase in
the amount of NO metabolites leaving an organ was the heart. This
indicates that at rest, there is a significant production of NO in the
coronary circulation. In addition, to determine whether the measurement
of nitrate in a single sample of venous blood reflected the production
of NO by an organ, we performed a regression analysis of the
arterial-venous difference of nitrate and the measured venous plasma
level. There was a significant correlation in the coronary circulation
and in the pulmonary circulation but not for mixed-venous or peripheral
venous blood. Thus, simple measures of venous plasma nitrate are not
good predictors of NO production by either skeletal muscle or the whole
circulation.
The calculated half-life was 230 minutes, or 3.8 hours. This means that it would take 19 hours, 5 half-lives, to totally clear an elevated nitrate level from plasma. In addition, because the clearance rate is slow—for comparison, the half-life of a filtered substance by the kidney is 20 minutes—there must be a number of mechanisms involved in the handling of nitrate once it is secreted into the plasma. Greene and Hiatt,18 19 in 1953 and 1954, found that nitrate is primarily cleared by the kidney but that nitrate is reabsorbed preferentially instead of chloride in the renal tubules. Thus, at least two renal mechanisms, filtration and reabsorption, will determine not only the plasma half-life of nitrate but also the amount of nitrate appearing in the urine.
Because of the long half-life, especially if there is a repeated stimulus for NO production, nitrate will accumulate not only in the venous blood but also in arterial blood. Under these conditions, the arterial-venous nitrate difference, the real measure of production by the whole body or a specific organ, cannot possibly be estimated from a single venous sample. In disease states, eg, heart failure, in which renal function is altered and the filtration rate and/or tubular reabsorptive mechanisms of nitrate changed, the plasma half-life may even increase. Thus, the comparison of plasma venous nitrate in normal patients and patients with heart failure may not reflect an increased production of nitrate but rather an altered excretion.
Even measurement of the arterial-venous nitrate difference across an organ, if the stimulus for nitrate production is long, may not reflect the molar production rate of NO. We calculated the volume of distribution of nitrate in the conscious dog and found that it most closely approximated the extracellular fluid volume, not the plasma volume. Therefore, since the ratio of the extracellular volume to plasma volume is approximately 4:1 to 6:1 (21.5% of the body weight/4% of the body weight), the measurement of the concentration of nitrate in plasma will grossly underestimate the real production rate of NO. For instance, if one assumes a plasma volume in a 70-kg man of 3 L and measures a nitrate concentration of 10 µmol/L, the actual amount of nitrate is 30 µmol. If, on the other hand, the 10-µmol/L concentration of nitrate is distributed in the extracellular volume (21% of 70 kg), the volume of distribution of nitrate is 14.7 L and the total nitrate is 147 µmol. Furthermore, should the extracellular volume change in a disease state such as heart failure, in which extracellular volume is markedly increased, the volume of distribution may be larger, making the measurement of plasma nitrate an even more substantial underestimate of NO formation.
In a recent study by Winlaw et al,15 there was a significant increase in venous plasma nitrate, from 24.6 to 62.4 µmol/L, in patients with heart failure. The authors then concluded that there is an increase in NO production in heart failure and further that this may reflect an increase in the production of NO by the heart, a contention supported by our own data. The 3-fold increase in plasma nitrate is in fact an estimate of changes in nitrate in the plasma volume, not extracellular water, as we report. Thus, the 3-fold increase in plasma nitrate is really a 15-fold increase in nitrate production, when one considers that the extracellular volume is 4 to 6 times the plasma volume. In addition, since the coronary blood flow is only 5% of cardiac output (a comparable value is also assumed in heart failure), the amount of nitrate produced by the heart would have to be 20-fold higher than measured in venous plasma. Taken together, this would indicate that nitrate production by the heart must increase by 300-fold to account for the increase in plasma nitrate measured in that study.15 This also assumes that the volume of distribution has not changed in heart failure, when, as evidenced by peripheral edema, the extravascular volume has probably increased. Since renal function is most assuredly depressed after the development of heart failure, a major component of the increased plasma nitrate is most likely the decreased filtration and increased reabsorption and not an enormous production by the heart. In one study that measured the activity of the inducible form of NO synthase in samples of human cardiomyopathic hearts, there was a 10-fold increase in NO production above that produced by the constitutive enzyme.26 This is much less than the 300-fold increase by the heart that would be necessary to account for a 3-fold increase in circulating nitrate. Furthermore, the reported concentration of nitrate varies greatly, from 20 µmol/L15 to 58 to 97 µmol/L14 to 26 µmol/L13 in normal people. Thus, a great deal of caution should be used when estimating the magnitude, let alone the source, of elevated plasma nitrate from samples of venous blood in normal or disease states.
Some attention has been paid to the origin of nitrate in body fluids dating back to a report by Mitchell et al in 1916,27 since nitrite is used as a food preservative and certain reactive nitrogen oxide species are potent carcinogens. Nitrate can be ingested in food, primarily in vegetables, or nitrite, a meat preservative, can be converted to nitrate in plasma. Nitrate is also consumed in water and can be formed from inhaled nitrogen dioxide in air.28 Green et al28 estimated the relative contributions of these sources and the amount of nitrate that is endogenously synthesized in humans, perhaps now as an index of NO production. These authors estimate that approximately half of the nitrate excreted in humans per 24 hours is endogenous and half is from other sources. Since the clearance rate of nitrate is estimated at only 20 mL/min13 or 26 mL/min,28 nitrate must be filtered and then reabsorbed in the ascending limb of the distal tubule in the kidney. At very high, millimolar concentrations of nitrate in plasma, nitrate may also be secreted by the kidney,29 as indicated by an increase in the nitrate/creatine ratio in the urine. Some estimates also indicate that only 40% to 70% of nitrate is excreted in the urine30 and the remainder in the feces,28 predictably a slow and variable process. All of these considerations make extrapolations of the production of NO by simple measures of plasma venous nitrate or both nitrite and nitrate precarious.
Intravenous infusion of nitrate in our study resulted in increases in arterial pressure and urine flow rate. By substitution into the linear regression equation for the changes in arterial pressure versus plasma nitrate, an increase in plasma nitrate into the millimolar range would be necessary to increase arterial pressure to any significant degree. Although a substantial diuresis occurred during nitrate infusion (urine flow rate increasing from approximately 0.5 to 5 mL/min), this is most likely a pharmacological effect and not a response that might occur at physiological concentrations of nitrate.
In summary, our studies in conscious dogs have shown that nitrate is a measure of NO production in plasma and that, although nitrite is also produced, nitrite is constant and by itself is not an index of NO formation in vivo. There is a net production of nitrate across the coronary circulation but not across any other vascular bed studied. The half-life for nitrate in plasma is relatively long, approximately 3.8 hours, and the volume of distribution is greater than the plasma volume, the calculated volume more closely approximating the extracellular fluid volume. The long half-life and large volume of distribution have to be taken into consideration in evaluation of changes in plasma nitrate as an index of NO formation in studies in both patients and animals. Since the primary mechanism for clearance of nitrate is renal, changes in plasma nitrate concentration in diseases in which renal function is altered or the extracellular volume is altered, which both occur in heart failure, for instance, may not reflect the production of NO but rather altered metabolism or volume of distribution.
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Acknowledgments |
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This study was supported by grants PO-1-43023, HL-31069, HL-50142, and HL-53053 from the National Heart, Lung, and Blood Institute.
Received October 3, 1994; revision received December 15, 1994; accepted December 27, 1994.
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