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Articles |
ur
Laboratory of Anesthesia, Departments of Anesthesia,
1 Biochemistry, and
2 Medicine, Hôpital Notre-Dame, Université de Montréal, 1560 Sherbrooke St. East, Montréal, QC, Canada H2L 4M1.
a Author for correspondence. Fax 514-896-4754; e-mail blaiseg{at}ere.umontreal.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: indexing terms: endothelium-derived relaxing factor • nitric oxide synthase • free radical • vasodilation • inflammation • thrombosis • immunology • neurotransmission
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Measurement of NO concentration in biological systems is a challenging analytical problem (3)(4). The chemiluminescence detector-based method for trace NO2- and (or) NO3- in aqueous samples was first reported by Cox (5) and was later widely applied (6)(7)(8). This earliest and most commonly applied method is based on the conversion of NO2- to NO at room temperature by an acetic acid–sodium iodide (NaI) mixture. Ammonium molybdate [Mo(VI)] with ferrous ammonium sulfate [Fe(II)] in hot, 50% concentrated sulfuric acid was used for the reduction of both NO2- and NO3- to NO. NO3- was then determined as the difference of results obtained by the two methods. Vanadium (II) [V(II)] was mentioned as a possible reducing agent in the initial work by Cox (5), who reported that it reduces NO3- to NO. Later work by Braman and Hendrix (9) indicated that it is V(III), not V(II), that reduces NO3- to NO. Stronger reducing agents such as chromium (II) [Cr(II)] and titanium (III) [Ti(III)] could also reduce NO3- to NO (10)(11). However, a systematic evaluation of different reducing agents and temperature conditions for the conversion of NO2- and NO3- to NO has not been performed. We compared the efficiency of V(III), Mo(VI) + Fe(II), NaI, Ti(III), and Cr(III) at different temperatures (20, 30, ... 90 °C) for the conversion of NO2- and (or) NO3- to NO. We also evaluated the recovery of NO2- and NO3- from plasmas of pig and of dog.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). We did not detect any NO2- or
NO3- in the blanks used in this study.
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reagents
NO gas and nitrogen were purchased from Air Liquide (Canada),
Montréal, QC, Canada. Vanadium trichloride, and sodium nitrite
and nitrate were purchased from Aldrich Chemical Co. (Milwaukee, WI).
Chromium trichloride, titanium trichloride, ferrous ammonium sulfate,
ammonium molybdate, sodium iodide, and other reagents were purchased
from Fisher Scientific. All were reagent-grade quality and used without
further purification. High-purity distilled water was used in the
preparation of all solutions.
recovery from aqueous solution
V(III), Mo(VI) + Fe(II), NaI, Ti(III), and Cr(II) have been
reported as reducing agents for the conversion of
NO2- and (or)
NO3- to NO
(5)(6)(8)(9)(10)(11)(12)(13). Except for Cr(II),
for which we substituted Cr(III) with its three valences, all of these
reducing agents were prepared as reported. Cr(III) was prepared in the
same conditions as the other two reducing agents with three valences.
Samples of inorganic NO2- (NaNO2)
or NO3- (NaNO3) in 250 µL of
water (100, 200, ... 500 pmol) were injected into the microreaction
purge vessel containing 5 mL of reducing agent solution, and the
quantity of produced NO was measured after conversion by each reducing
agent at 20 °C for NO2- and at 80 °C for
NO3-. The effect of temperature on the
conversion of 400 pmol of NO2- and (or)
NO3- (in a volume of 250 µL) to NO with each
reducing agent was determined by changing the temperature scale from
20, 30, ... to 90 °C. The chemiluminescence analyzer was
calibrated by injecting known amounts of NO gas (100, 200, ... 500
pmol) through the microreaction purge vessel heated at different
temperatures in the absence of reducing agent solution. Data were
collected as area under the curve response from baseline to baseline
and divided by the mean standard response of NO gas for each
concentration. From this we obtained a recovery factor (R) expressed in
percentage. Serial measurements can be performed for each
NO2- or NO3-
concentration without changing the reducing agent solution in the
microreaction purge vessel, since the volume of added samples is very
small compared with the volume of the reducing agent solution.
recovery from plasma
We added known amounts (100, 200, ... 500 pmol) of inorganic
NO2- or NO3- in
0.1 mL of pig and dog plasma and then measured baseline amounts of
NO2- and (or) NO3- to
evaluate their recovery. Excessive foaming in the microreaction purge
vessel caused by plasma proteins interfered with the reduction process.
Therefore, all determinations in plasma samples were performed after
deproteinization. Plasmas were diluted 10-fold with distilled water and
deproteinized by addition of 1/20th volume of zinc sulfate to a
final concentration of 15 g/L. After centrifugation at 1000g
for 15 min, 0.1 mL of supernatant was applied to the microreaction
purge vessel containing NaI solution at 30 °C for the conversion of
NO2- to NO, or V(III) solution at 80 °C for
the conversion of NO3- +
NO2- to NO. Samples of
NO2- or NO3- added in
plasma were compared with those prepared in distilled water in the same
condition. This method only detects NO2- and
NO3- that can readily pass into the gas phase
to react with the O3 in the microreaction purge vessel.
Thus, any of the NO formed in vivo that would react with thiol groups
in low-molecular-mass compounds is not detected here.
statistical analysis
Unless otherwise stated, all data are expressed as the mean
± SE of R. Global mean indicates the mean of all values of R
calculated for each of the five agents with all concentrations of
inorganic NO2- or
NO3- used. The statistical analyses were done
with the SAS statistical analysis program. The significance level was
set at 0.05. The analysis of variance (GLM procedure) was used to
compare values of global means (P1) for the
five reducing agents. The analysis was repeated for each amount of
samples used (100–500 pmol). The post hoc analysis between the five
reducing agents was realized by using the Student–Newman–Keuls (SNK)
tests. The effect of temperature and agents on R values was analyzed by
using the two-way analysis of variance (P2). The
SNK post hoc analysis was used to compare R values for each agent at
different temperatures and R values for different agents at the same
temperature. The optimal reduction temperature(s) was (were) determined
as the temperature(s) with the highest R value significantly different
from the other R values. Separate paired t-tests were used
for dog and pig plasma when the recovery of
NO2- was compared with that of
NO3- (P3).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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.
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effect of temperature on conversion of 400 pmol of
no2- to no
Temperature had no effect on the detection of NO gas by
chemiluminescence (data not shown; P1 = 0.63).
Recovery of NO from NO2-
(P2 = 0.01) by the five reducing agents was
affected by temperature (Fig. 2
). For NaI, the lowest R value (89.1%) was obtained at 20 °C
(SNK P <0.05) and no significant difference was found
between the other temperature values. For other agents, the optimal
reduction temperature was Mo(VI) + Fe(II) 50–60 °C, V(III)
60–80 °C, Cr(III) 20–70 °C, and Ti(III) 20–60 °C. The
comparison between agents showed that the R values with NaI and Mo(VI)
+ Fe(II) were higher than the R values of the three other agents at
50–60 °C (SNK P <0.05).
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effect of different reducing agents on conversion of
no3- to no at 80 °C
The recovery of NO from different amounts of
NO3- (100–500 pmol) was <1.7% when
using NaI and Cr(III) as reducing agents. Often, no conversion was
detected. The global mean of the R value for Ti(III) was 82.3% ±
1.8%, whereas those for V(III) and Mo(VI) + Fe(II) were respectively
105.8% ± 1.6% and 101.1% ± 2.8%. The R values obtained for each
amount of NO3- are summarized in Table 2
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effect of temperature on conversion of 400 pmol of
no3- to no
Temperature affected the recovery of NO from
NO3- by V(III), Mo(VI) + Fe(II), and
Ti(III) (P2 = 0.0001; Fig. 3
). The optimal reduction temperature was 80–90 °C for V(III)
and 70–90 °C for Mo(VI)+Fe(II) and Ti(III). Temperature did not
affect the low recovery (<1.7% at any temperature tested) of NO from
NO3- by NaI or Cr(III).
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recovery from plasma
For pig plasma, the recovery of NO2-
was 96.4% ± 1.9% (n = 20) and the recovery of
NO3- was 104.3% ± 4.9% (n = 20) over
the entire concentration range tested (100–500 pmol). There was no
significant difference between the recovery of
NO2- and NO3-
(P3 = 0.21). For dog plasma, the recovery of
NO2- was 89.6% ± 2.0% (n = 20) and the
recovery of NO3- was 107.4% ± 2.4% (n
= 20) over the entire concentration range tested (100–500 pmol). The
difference between the recovery of NO2- and
NO3- was significant
(P3 = 0.0008).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Determination of NO in itself is difficult because of its free-radical nature and short half-life. NO reacts rapidly with oxygen to form NO2- and with superoxide or with oxyhemoglobin to form NO3-. In most cell culture systems (2), NO will be oxidized primarily to NO2-, whereas in animal models and human samples, NO is oxidized both to NO2- and NO3-. Nitrite and nitrate are both stable in frozen plasma for at least 1 year (2). Therefore, determination of the stable end products of the NO radical is most often used to measure its concentration. NO3- is the major metabolite of NO in blood (2); thus, determination of NO2- alone as a marker for NO concentration is meaningless, even if some previous studies had reported NO2- as the major byproduct of NO in blood (14). The availability of a quantitative assay for both NO2- and NO3- can facilitate further elucidation of some of the physiological, pathophysiological, pharmacological, and therapeutic roles of NO.
Compared with other analytical methods (2)(3)(4) (e.g., spectrophotometry, electron paramagnetic resonance, gas or liquid chromatography, mass spectrometry), chemiluminescence is highly sensitive, selective, and accurate for NO2- and (or) NO3-, especially at the low concentrations in complex matrices found in water, food, and biological fluids. The key point in this procedure is to choose the appropriate reducing agent/temperature combination to selectively and completely reduce NO2- or NO3- to NO.
Several reducing agents have been tested for the reduction of NO2- and (or) NO3- to NO, such as NaI for the conversion of NO2- to NO, and V(III), Mo(VI) + Fe(II), Ti(III), and Cr(II) for the conversion of NO3- to NO (5)(9)(10)(11)(12)(13). NO2- can be reduced to NO by using most reducing agents at room temperature, whereas conversion of NO3- to NO requires both a strong reducing agent and high temperature. V(III) and Ti(III) with three valences can reduce most of NO3- to NO at high temperature. We compared the efficiency of Cr(III), also with three valences, with V(III) and Ti(III) for the conversion of NO2- or NO3- to NO.
Our work revealed that the five reducing agents have a similar efficiency for the conversion of NO2- to NO at 20 °C, with a slight advantage for Mo(VI) + Fe(II) and Ti(III) over the other three agents. The recovery of NO from NO2- was almost complete, compared with the known amount of NO gas standard [lower R value: 86.2% ± 1.6% for Cr(III) for the recovery of 100 pmol of NO2-].
V(III) and Mo(VI)+Fe(II) were equally efficient in converting NO3- to NO at 80 °C, and recovery of NO was nearly complete. However, recovery with Ti(III) was lower. NaI and Cr(III) were unable to reduce NO3- to NO, as only trace amounts of NO were recovered from NO3- regardless of the amount of NO3- and the temperature used. NaI and Cr(III) can thus be considered selective for reducing NO2- to NO. To our knowledge, this is the first report to show that Cr(III) can selectively reduce NO2- to NO. Enzymatic reduction of NO3- by using an immobilized Escherichia coli nitrate reductase column (15) converts ~30% of NO3- to NO2-. Another assay based on the coupled oxidation of NADPH during the enzymatic conversion of NO3- to NO2- by Aspergillus nitrate reductase only yields ~64% of serum NO3- to NO2- and is unsatisfactory for NO3- analysis in serum samples. Furthermore, though many reports claim a possible recovery of 100%, commercial nitrate reductases are rather expensive (16). Powerful chemical reducing agents such as V(III), Mo(VI) + Fe(II), and to a lesser degree Ti(III) are more efficient than nitrate reductases for converting NO3- to NO2- in biological samples.
The temperature affected the conversion of both NO2- and NO3- to NO, as the reduction process is facilitated and more rapid at higher temperatures. This is particularly true for the conversion of NO3- to NO. However, increasing temperature had several technical drawbacks on the conversion of NO2- and NO3- to NO. The first is that the measurement of NO by chemiluminescence is influenced by humidity (3)(4). As temperature increased, the heat evaporated more water, which quenched NO2 produced by the O3 reaction. The second problem encountered is that the reducing solution has a tendency to move from the microreaction purge vessel to the condenser and even to the NO analyzer itself at high temperatures. We therefore propose that 60 °C would be the really appropriate temperature for converting NO2- to NO by these five reducing agents. In the case of NaI, the most efficient NO2- reducing agent at any temperature, increasing the temperature had the undesirable effect of increasing the variability of the results (larger SE) and lowering the reproducibility, because of the above-mentioned reasons. The same situation was observed for the conversion of NO3- to NO. It would thus be very useful to find and select a reducing agent that can reduce NO3- to NO at low temperatures.
It has been suggested that strong reducing agents, such as V(III), can
be used at different temperatures to achieve different goals: at low
temperatures for the conversion of NO2-
to NO, and at high temperatures for the conversion of
NO2- + NO3- to NO.
NO3- would then be determined by the
difference between analysis of the same sample by both assays. Fig. 3
shows that V(III), Mo(VI) + Fe(II), and Ti(III) can also reduce
NO3- to NO at low temperature, albeit at a low
degree. Use of only two different temperatures cycling for a strong
reducing agent to selectively reduce NO2- and
(or) NO3- to NO can be a cause of
overestimated NO2- and underestimated
NO3- measurements.
Our results indicate that the most accurate procedure is to use NaI or Cr(III) as a reducing agent to selectively convert NO2- to NO at low temperatures and then use a strong reducing agent to convert all NO2- + NO3- to NO at 80 °C or 90 °C.
Proteins contained in most biological samples can cause excessive foaming in the microreaction purge vessel and interfere with the reduction process. Deproteinization is therefore essential for the analysis of such samples. Investigating the recovery of both NO2- and NO3- in biological samples with particular attention given to the reproducibility of the assay and the occurrence of artifacts is important. In this study, recovery of NO2- and NO3- in deproteinized plasma was 93.0% ± 1.6% and 105.9% ± 2.7% respectively. Although the recoveries of NO2- and NO3- were similar and complete for the pig plasma, we discovered a small but significant difference in the case of the dog plasma. The residual protein environment in the dog plasma seemed to interfere more with NO2- than with NO3-. Though part of the NO formed in vivo may react with thiol groups in low-molecular-mass compounds, this method does not detect these low-molecular-mass nitroso compounds, as NO must be in the gas state to react with the O3 in the reaction chamber.
Compared with the same amount of NO gas, V(III), Mo(VI) + Fe(II), NaI, Ti(III), and Cr(III) are similarly efficient reducing agents for the conversion of NO2- to NO at 20 °C. V(III) and Mo(VI) + Fe(II) can also completely reduce NO3- to NO at high temperatures. However, Cr(III) and NaI were unable to convert NO3- to NO. Cr(III) and NaI can specifically reduce NO2- to NO. We recommend the use of NaI or Cr(III) at room temperature to selectively and completely reduce NO2- and the use of V(III) or Mo(VI) + Fe(II) at 80–90 °C to reduce NO2- + NO3- to NO. Recovery of both NO2- and NO3- in experimental animal plasma was reproducible and near quantitative, albeit to a lesser degree in the case of the dog plasma. These results also highlight the need for a relatively large-scale study with human subjects to establish proper baseline measurements for clinical assays of plasma nitrite and nitrate concentrations.
We estimate that a properly organized clinical laboratory could process ~30 samples/h for NO2 measurements and ~15 samples/h for the measurement of NO3 concentrations.
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Acknowledgments |
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References |
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