half-life of nitric oxide in aqueous solutions with and without haemoglobin

7/31/2019 Half-Life of Nitric Oxide in Aqueous Solutions With and Without Haemoglobin

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Physiol. Meas. 17 (1996) 267277. Printed in the UK

Half-life of nitric oxide in aqueous solutions with and

without haemoglobin

T S Hakim, K Sugimori, E M Camporesi and G Anderson

Departments of Surgery, Anesthesiology and Medicine, SUNY Health Science Center, 750 East

Adams Street, Syracuse, NY 13210, USA

Received 16 January 1996, in final form 7 August 1996

Abstract. Nitric oxide (NO) has been linked to many regulatory functions in mammalian cells.

Studies of NO release are hampered by the short half-life of the molecule. In the blood, NO

disappears within seconds because it binds avidly with haemoglobin (Hb). The relationship

between Hb concentration and NO disappearance, however, has not been described. In this

study we utilized an amperometric NO sensor (WPI, Sarasota, FL) to monitor continuously

the disappearance of NO from an aqueous solution when Hb (free or as red blood cells)was added. The calibration and linearity of the NO sensor was checked frequently using

a chemical reaction to generate a known concentration of NO. An aliquot of NO solution

(prepared from authentic gas) was added to a glass beaker containing 20 ml saline to generate NO

concentration of approximately 1200 nM. Under our experimental conditions (PO2 = 40 mmHg),

NO concentration fell slowly over 20 min with a half-life of 445 s. However, when haemoglobin

was added, NO disappeared rapidly in proportion to Hb concentration. The results suggest that

rapid binding of NO to Hb occurs in a 4:1 ratio. The maximum rate constant of NO disappearance

due to binding with Hb was 2 105 M1 s1. The 4:1 binding ratio between NO:Hb may be

used as a tool to quantitate NO release in some biological assays. The study supports the notion

that NO acts as an autocoid because it disappears rapidly in the presence of Hb and is not likely

to act as a circulating humoral substance. The NO sensor was useful for monitoring of NO

concentration in Hb free solutions, but its response time limits its use in blood.

Keywords: nitric oxide, haemoglobin, rate constant, temperature, meter

1. Introduction

Endogenously released nitric oxide (NO) has a short biological half-life of 5 s or less (Archer

1993, Bates 1992, Nathan 1992). The reaction rate in aqueous solutions of NO with oxygen

and with haemoglobin (Hb) follows a second-order kinetics, and therefore the rate of NO

disappearance is proportional to the square of NO concentration (Ignarro et al 1993). At high

concentrations of NO (300 M) in aqueous solution, a half-life of< 1 s has been reported,

while at lower concentration of 0.011 M (compatible with the biological concentration),

the half-life has been reported to be 500 s (Wink et al 1993). Clancy et al (1990) found

that 0.5 M NO in aqueous solutions equilibrated with room air has a half-life of 1015 s,

but a much longer half-life at low PO2

of 14 mmHg. In the absence of haemoglobin and

oxygen, NO in solution is stable for several days (Archer 1993). The rapid reaction of NO

with Hb has been used extensively as a tool to scavenge NO and to inhibit its action as a

vasodilator (Ignarro et al 1993). The half-life of NO has usually been evaluated indirectly

by measuring the products of NO such as nitrosohaemoglobin (NOHb), methaemoglobin

0967-3334/96/040267+11$19.50 c 1996 IOP Publishing Ltd 267


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268 T S Hakim et al

(metHb), or NO2/NO3 concentration (Archer 1993, Bates 1992, Ignarro et al 1993, Feelisch

and Noack 1987). Because the exact steps which lead to NO degradation are not clear, the

reported half-life may reflect variables other than NO reaction. This study presents data

from continuous monitoring of NO at biological concentrations (< 1.5 M) in aqueous

solution, and in the presence of haemoglobin at low molar concentrations.

2. Materials and methods

2.1. NO meter calibration

NO was measured with a commercially available NO meter (Iso-NO, World Precision

Instrument, Inc., Sarasota, FL). Details of a prototype electrode design has been described

(Shibuki 1992) and results with this specific meter have been published (Tsukahara et al

1993). The sensor probe housing contains an electrolyte solution and is covered with a gas-

permeable membrane. Similar to the Clark-type electrode for oxygen, NO diffusing through

the membrane is oxidized at the working platinum electrode resulting in an electrical current.

The probe polarographically measures the concentration of NO gas in aqueous solutions.

The NO concentration is displayed as a redox current. The electrical current is proportional

to the rate of diffusion through the membrane, which is, in turn, proportional to the NO

concentration at the membranes outer surface.Probe calibration was accomplished with KNO2 (0.05 mM) as a generator of NO in a

solution containing 0.1 M KI, 0.1 M H2SO4, 0.14 M K2SO4 mixture (as recommended by

the manufacturer) based on the following stoichiometric reaction:

2KNO2 + 2KI+ 2H2SO4 + 2K2SO4 2NO+ I2 + 2H2O+ 4K2SO4.

A 50 cm3 glass beaker containing 20 ml of the standard chemical mixture was placed on a

magnetic stirrer and kept at constant temperature (either 25 C or 37 C). The temperature in

the solution was monitored (Omega 2000, Stamford, CT). The solution was deoxygenated

by purging with nitrogen for 15 min. The NO probe was inserted vertically into the beaker

so that the tip of the electrode was 5 mm under the surface. The output signals from the

NO meter in picoamperes (pA) and from the thermistor were directed to a data acquisition

system (Biopac MP100), sampled at 2 Hz, and stored on a computer (figure 1). When the

signals reached a stable level, purging with N2 was stopped by pulling the tube to abovethe surface of the solution, in order to maintain low oxygen in the beaker. Small aliquots of

0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 ml of KNO2 solution were added cumulatively to the beaker

to generate increasing levels of NO concentration. These aliquots of KNO2 produced step

changes of 125, 248, 491, 726, 950 and 1160 nM NO in the beaker respectively and step

changes in meter output reaching maximum in approximately 40 s. The step changes in NO

concentration were plotted against the maximum step changes in NO meter output (in pA)

after addition of each aliquot, and the slope was calculated by linear fit regression analysis.

The slope (nM pA1) was used as a conversion factor to convert changes in pA output to

NO concentration. Calibration of the probe was performed daily.

2.2. NO solution preparation

Standard NO solution was prepared by dissolving 0.5 ml of authentic NO gas (99% pure,

MG gas) in 50 cm3 of deoxygenated 0.9% saline contained in an airtight glass syringe.

The saline was deoxygenated by purging vigorously with nitrogen for 1 h. This procedure

usually lowers the partial pressure of oxygen from 150 to < 35 mmHg. A 50 cm3 glass


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Nitric oxide measurement 269

Figure 1. A diagram of the experimental set-up used for monitoring nitric oxide concentration.

syringe was coated with stopcock grease, flushed twice with the deoxygenated saline, filled

with deoxygenated saline, and sealed with a self-sealing rubber cup. All the gas in the

syringe was expelled through a 22-gauge needle. NO gas (0.5 ml) was withdrawn with a

1 cm3 plastic syringe after flushing twice with NO, and introduced slowly via a 22-gauge

needle into the sealed glass syringe. The glass syringe was vibrated vigorously to dissolve

the NO gas. Using this procedure a small bubble (< 0.1 ml) usually remained undissolved in

the syringe. Thereafter, the glass syringe was always kept sealed and gasless. Withdrawinga sample from the glass syringe with a 1 ml syringe and a 22-gauge needle was always done

while pressuring the glass syringe to minimize diffusion of room air into the glass syringe.

The NO concentration remained relatively stable over a two-week period. Based on the

gas equation PV = nRT, where R is the gas constant (0.08 206 l atm K1 mol1), and

assuming a barometric pressure of 750 mmHg (0.99 atm), a temperature of 25 C (298 K)

and 100% NO, the number of moles in 0.5 ml (0.0005 l) would be 20.2 106. When

dissolved in 50 ml saline, the final concentration would be expected to be 404 M. As will

be discussed in the results, however, the concentration was usually < 200 M.

2.3. Haemoglobin preparation

Heparinized blood was withdrawn from Sprague-Dawley rats. Half of the blood was

kept as whole blood and the other half was centrifuged for 20 min at 3000 rpm. The

plasma and buffy coat were discarded from the centrifuged portion. The red blood

cells (RBCs) were washed twice with saline, centrifuged and the supernatant discarded.

Distilled water was then added to the packed RBCs, stirred gently and allowed to sit


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270 T S Hakim et al

for 10 min for haemolysis. The haemolysed blood was centrifuged again for 20 min

and the supernatant containing free haemoglobin was saved. The total haemoglobin in

the whole blood and in the haemoglobin solution was measured using a haemoximeter

(OSM3, Copenhagen). Eight serial dilutions of blood and Hb (ranging from 0.02 g% to

5.4 g%) were prepared and allowed to equilibrate with room air for 1 h before they were

used.

2.4. Experimental protocol

Nine 50 cm3 glass beakers were filled with 20 ml sterile 0.9% saline and purged gently

with nitrogen for 15 min (same set-up as in figure 1). When the NO meter output was

stable, the nitrogen purging in the beaker was stopped but nitrogen continued to flow into

the beaker. NO solution (0.2 ml) was withdrawn from the stock syringe and injected into

the beaker. The NO concentration viewed on the computer monitor rose immediately and

reached a peak after 10 s. At peak NO concentration, 0.2 ml saline (Hb = 0) was added to

the first beaker. The NO concentration declined slowly over the next 20 min. After the NO

concentration declined by more than 60% of peak, the NO probe and thermometer were

removed, rinsed thoroughly with distilled water and placed in a second beaker containing

deoxygenated saline. When the signal became stable, nitrogen purging was stopped asbefore, and 0.2 ml of the NO was added. At peak NO concentration, 0.2 ml of RBC

suspension (Hb = 0.02 g%) was added to the second beaker and NO decay was observed.

This procedure was repeated in seven more beakers. To every beaker, 0.2 ml of NO solution

was added and followed by 0.2 ml of RBC suspension with increasing concentration of

RBCs. The same procedure was repeated in nine other beakers, but instead of adding RBC

suspension, free Hb solution (0.2 ml) was added. The final concentration of Hb in each

beaker is reported in the results. All the data were stored on the computer for off-line

analysis.

Figure 2. Analysis of the nitric oxide tracings. Panel A shows appearance and decay of nitric

oxide after addition of 0.2 ml of nitric oxide solution and after addition 0.2 ml saline containing

no haemoglobin. Panel B shows the decay of nitric oxide after addition of haemoglobin (Hb).

Analysis of the tracings is described in the text.


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2.5. Analysis

Figure 2(a) illustrates a typical recording of NO concentration after addition of 0.2 ml NO

solution and its decay after adding 0.2 ml saline (Hb = 0). Decay of NO was estimated

from the half-life (T1/2), the time required for the NO concentration to decrease by 50%.

Figure 2(b) illustrates a typical tracing of NO concentration in a beaker into which Hb was

added at peak NO concentration. A second-order chemical reaction between NO and Hb

was assumed to apply. The reciprocal of the NO concentration versus time was plotted andthe slope of a linear fit (M1 s1) was calculated as the rate constant, K , for each beaker.

The data used for this analysis were limited to 30 s (60 data points) after Hb was added. The

amount of NO which disappeared rapidly after addition of Hb (NO), was also estimated

(figure 2(b)). This was, in most instances, easily identifiable as the bend point between the

rapid and slow phases of NO disappearance. The bend point was approximated by drawing

two straight lines through the two phases. The rapid fall in NO concentration (NO) was

assumed to be due to rapid binding of NO with haemoglobin.

Figure 3. A typical tracing of the nitric oxide meter calibration. The relationship was linear in

this range and the slope was used as the calibration factor.

3. Results

3.1. NO meter calibration

Typical tracings of a calibration procedure are shown in figure 3. The relationship between

NO concentration generated by the chemical reaction and the meter output was linear with

a correlation coefficient of> 0.99, but was variable on different days as previously reported

(Tsukahara et al 1993). The slope of the calibration (nM pA1) on a given day was used


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272 T S Hakim et al

to convert the NO meter output to NO concentration. The slopes at 37 and 25 C were

1.8 0.49 and 3.63 0.44 nM pA1 (meanSD, n = 5) respectively. The NO meter was

more sensitive at 37 C than at 25 C.

Figure 4. Appearance and decay of nitric oxide concentrations with time in one set of beakers.

3.2. NO decay

Figure 4 illustrates segments from a typical set of tracings recorded after addition of NO and

haemoglobin to the reaction beakers. Only five tracings are shown because the tracings from

the last four beakers with the highest Hb concentrations were nearly identical. The addition

of 0.2 ml NO solution produced consistent responses of 1300 nM in these tracings. ThePO2 in the solution (after purging with nitrogen) was 40 mmHg (Radiometer, ABL II blood

gas analyser). Figure 5 illustrates in more detail the changes in NO concentration during

a 2 min period after addition of haemoglobin in one set of nine beakers. The tracings are

superimposed on each other to allow comparison. Typically the shapes of the tracings in the

three or four beakers with the highest haemoglobin concentration did not differ significantly.

The half-life of NO decay in the first beaker (Hb = 0) under these conditions averaged

445.477.3 s (meanSD, n = 10). When Hb was added, NO fell rapidly. At the highest

Hb concentration, NO fell to 50% in 11.52.1 s (meanSD, n = 10). The combined results

from all experiments with red blood cells (at 25 C and 37 C) and with free haemoglobin

(at 25 C) are illustrated in figures 6 and 7. There was no discernible difference between

effect of red blood cells and free haemoglobin or between the values at 25 C or 37 C.

Figure 6 illustrates the relationship between the final concentration of haemoglobin in the

beaker and the rapid loss of NO from the beaker (NO). NO increased as haemoglobin

concentration rose but plateaued after the haemoglobin concentration exceeded 310 nM.

The highest concentration of haemoglobin used in this experiment was 8000 nM but the

maximum effect on rapid NO disappearance was accomplished with 310 nM. The data


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Figure 5. Decay of nitric oxide immediately after addition of haemoglobin. Beakers 6, 7, 8

and 9 are clustered together. The concentrations of haemoglobin which were added to the nine

beakers were 0 (1), 31 (2), 78 (3), 155 (4), 310 (5), 620 (6), 1550 (7), 3100 (8) and 8370 (9)

nM respectively.

between 0 and 310 nM Hb were fitted by a linear regression to a straight line. The equation

of the line is shown in the figure with a slope of 3.97. This suggested that each molecule

of haemoglobin binds rapidly with four molecules of NO. Furthermore, increasing the

concentration of haemoglobin beyond four times the concentration of NO had no additional

effect on NO disappearance. The haemoglobin which was added to beakers 6, 7, 8 and 9

led to loss of8595% of NO very rapidly.

Figure 7 shows the changes in rate constant (K). The reciprocal of NO concentration

after haemoglobin addition was plotted versus time. The slope of the individual plots

(calculated by linear regression analysis for 20 s of data) was used as the rate constant

for a second-order kinetic reaction in each of the nine beakers. K increased gradually

as haemoglobin concentration was increased; reaching a maximum value of approximately

2.0 105 M1 s1. The inset in the same figure illustrates that maximum K was reached

when Hb/NO ratio approached 0.25. These results are consistent with the results in figure 6,

and corroborates the conclusion that maximum and fastest binding of NO to Hb is reached

when haem concentration was equal to NO concentration.

4. Discussion

The present study quantitates with continuous monitoring the rate of NO disappearance in

aqueous solution in the absence and in the presence of haemoglobin. The concentration

used in our study was comparable to biological concentrations. In aqueous solutions, NO

disappears due to oxidation to NO2 , and in the presence of Hb, NO binds to Hb to form


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274 T S Hakim et al

Figure 6. The relationship between the haemoglobin concentration and the nitric oxide which

disappeared rapidly (NO) from the solution. The linear regression fit was applied to the data

with haemoglobin concentrations < 500 nM. Circles = data obtained at 25 C with red blood

cells, diamonds = data obtained at 25 C with Hb, crosses = data obtained at 37 C with red

blood cells.

NOHB (Ignarro et al 1993). Oxidation of the NOHb with subsequent formation of metHb

and NO3 takes longer (Case et al 1979, Chiodi and Mohler 1985, Doyle and Hoekstra

1981). The amount of NO which disappeared rapidly was highly correlated to the amount

of Hb added until the haem concentration equalled that of NO concentration. Thereafter

excess Hb did not affect the disappearance markedly. The results suggest that, as expected,

each haem molecule binds rapidly to one molecule of NO. The reaction between Hb andNO was demonstrated by Hermann (1865) and confirmed by Gibson and Roughton (1957).

The characteristic binding of Hb to NO in a 1:4 ratio may be particularly useful in studies of

isolated vessels. By estimating the molar concentration of Hb which is required to scavenge

all the NO present in the solution and cause maximum increase in smooth muscle tone, it

is possible to approximate the concentration of NO which may be present in the solution.

Thus far, use of Hb to inhibit NO action has been made in a qualitative manner whereby

excess Hb is added to the bath to abolish the effect of NO (Ignarro and Kadowitz 1985).

Additionally, our study shows that the half-life of NO in an aqueous medium is

445 s, consistent with previously reported values of 500 s (Wink et al 1993) despite small

differences in pH, NO concentration and in temperature. Loss of NO concentration in this

study, when Hb is absent, however, may be primarily due to loss of NO from the solution

to the gas in the beaker because of the low partition coefficient of NO in solution. In the

presence of Hb, NO fell rapidly to 50% in 11.5 s. This is also consistent with previously

reported values of 1015 s for NO activity (Clancy et al 1990) or 30 s as reported by

Myers et al (1990). The maximum rate constant which was measured in this study was

2105 M1 s1. Although this is a rapid reaction compared to many enzymatic reactions, the


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Figure 7. The relationship between the rate constant and haemoglobin/nitric oxide concentration

ratio. The inset illustrates some of the data in more detail. Symbols are the same as in figure 6.

rate constant is smaller than the 1.2107 M1 s1 value reported by Gibson and Roughton

(1957). These authors have nevertheless mentioned that the measurement of rate constant

of this magnitude is close to the limits of their instrumentation and therefore may not beaccurate. It is possible that the meter which we used also has a limited response time. On the

one hand some of our results suggest that the response time of the NO probe was adequate

because the fastest reaction occurred when haem concentration equalled NO concentration.

On the other hand, the fact that the data at 37 C were not markedly different from those

at 25 C would suggest that the meter may have reached its maximum response time, and

perhaps was unable to detect reactions having a K value greater than 2 105 M1 s1.

In blood, NO activity is lost due to avid binding with Hb to form nitrosyl haemoglobin

(NOHb). It is not clear exactly what happens thereafter, or how quickly. NOHb is stable

in the absence of oxygen (Gibson and Roughton 1957, Case et al 1979, Chiodi and Mohler

1985, Kruszyna et al 1987) and is apparently stable at much lower temperatures, even in

the presence of oxygen (Cantilena et al 1992). In the presence of oxygen, the NOHb may

become oxidized to form MetHb and NO2/NO3, which are stable products and remain in the

blood for a few hours (Chiodi and Mohler 1985). Formation of metHb, in general, is thought

to occur in < 2 s (Archer 1993, Doyle and Hoekstra 1981). Although other factors may

influence the formation of NOHb, the nitrosylation of Hb remains very fast and accounts for

the loss of NO activity in the blood or in Hb solutions. The idea that NO acts as an autocoid


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276 T S Hakim et al

is strongly supported by our results. Some investigators suggested that deactivation of NO

in the blood may be linked to the saturation state of Hb with oxygen (Wennmalm et al

1992, Iwamoto and Morin 1993). The validity of such an idea is questionable because NO

has a great affinity with reduced Hb and would bind to it very rapidly regardless whether

the Hb is bound or unbound to oxygen (Ignarro et al 1993). In the interstitial space, NO

may have a longer half-life than in the vascular space because Hb is not present, but exactly

how long is not known. Stimulation of endothelial cells in culture causes the NO levelto rise and remain elevated for a few minutes (Tsukahara et al 1993). Whether the same

time course of NO deactivation occurs in vivo for the extravascular space is not known but

seems possible.

On the basis of the present results, deactivation of NO was very fast and was nearly

complete in a few seconds when excess Hb was present. Nevertheless oxygen saturation

affects the subsequent oxidation of NOHb to metHb (Chiodi and Mohler 1985). In

preliminary trials, we compared the rate of disappearance of NO in Hb-free solution,

and found only little difference between the half-life of NO in a solution containing low

PO2 = 40 mm Hg and that containing 150 mmHg. However, for consistency in the

present study, we always made the measurements after purging with nitrogen. In the intact

conditions and in whole blood, Hb concentration far exceeds the concentration of NO that

may be released.

NO action has been shown to be inhibited by Hb in a dose-related manner (Ignarro et al1987, Ignarro and Kadowitz 1985), but the time course was not usually considered. Adding

Hb to a vessel dilated by NO demonstrates the high speed by which Hb inhibits NO action:

for example in isolated vessels, acetylcholine-induced vasodilation is rapidly reversed by

Hb (Edwards et al 1986, Evans et al 1989). The rapidity with which NO activity disappears

due to binding with Hb has also been demonstrated in perfused lungs (Iwamoto and Morin

1993, Rich et al 1993, Rimar and Gillis 1993). NO administered into isolated perfused lungs

by inhalation or by infusion becomes deactivated during the transit through the vasculature

in the presence of low concentration of Hb (Rich et al 1993, Rimar and Gillis 1993). NO

inhalation studies in humans and in animals suggest that vasodilatory actions are limited to

the pulmonary vasculature (Rich et al 1993, Frostell et al 1991) suggesting that NO becomes

rapidly deactivated when it enters the circulation. Administration of NO by inhalation may

be detrimental in certain conditions because NO not only occupies some Hb molecules but

also increases the affinity of Hb to oxygen and thus may lead to reduction in oxygen supply(Kon et al 1977). Studies which have infused NO solution into intact animals have reported

minimal vascular response (Golino et al 1992) because NO will bind to Hb as quickly as

it is infused, and is not likely to influence remote sites. The results of the present study

suggest that the half-life of NO in the vascular space is approximately 11.4 s. The binding

of NO to Hb in a 1:4 ratio may be used for determining the NO concentration in Hb-free

preparations such as the isolated vessel.

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