INTRODUCTION

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NITRIC OXIDE (NO) metabolites in plasma [NO and NO (NOx)] have been used as an index of endothelial NO production in physiological and pathological conditions, especially in local circulation, based on the rapid oxidization of NO to NOx in the blood (2). In support of this notion, higher NOx concentrations have been reported in coronary sinus blood than in arterial blood in the conscious dog model (11, 16). In the human heart, however, no increase in NOx across the normal coronary circulation has been noted (7), and this finding has been supported by a recent study in the conscious dog model (14). Furthermore, a number of studies have shown a negative NOx balance in the coronary circulation in certain pathological conditions such as pacing-induced heart failure in conscious dogs (11), angina with significant organic stenosis, or with vasospasm in human subjects (5, 7). These findings seem unreasonable. Even when endothelial cell function is attenuated in some pathological conditions, NO would be released at levels that are detectable in the coronary circulation as an attenuated increment in NOx production. It has been also noted that addition of exogenous NO to whole blood (ex vivo) results in a smaller increase in plasma NO concentration than the estimated value based on volume of plasma as a sole compartment for distribution (7, 8, 11). From these observations, it is conceivable to consider that NOx may localize into other compartments apart from plasma under certain circumstances. As pointed out by Recchia et al. (11), red blood cells (RBC) may act as a second compartment for NOx in the blood. They recently showed that the NOx concentration in RBC is several times higher than in that in plasma and indicated that the PCO₂ and bicarbonate concentration of plasma may regulate NOx levels in both compartments (12). However, in their measurement procedure, they used an ultrafiltration unit that was known to be contaminated by NOx (6), leading to incorrect (higher) estimation of NOx concentration in RBC. Therefore, we have examined distribution of NOx in plasma and in RBC with a new technique, avoiding NOx contamination in the procedure. In addition, we examined whether dynamic fluctuations in potential regulatory factors (PCO₂ and bicarbonate concentration of plasma) induced by hyperventilation alter NOx concentration in the plasma and RBC of normal human subjects, whereas conclusions by Recchia et al. (12) were based on static blood samples collected from different vascular beds in conscious dogs.

	MATERIALS AND METHODS

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Subjects. To evaluate our system in determining the NOx concentration in blood (see below), venous blood was sampled from healthy volunteers (aged 25-50 yr of both sexes) from the laboratory staff of the Department of Pharmacology, Kanazawa Medical University. For the hyperventilation study, nine healthy male volunteers (age, 27-36 yr; height, 172 ± 1 cm; weight, 67 ± 3 kg) were recruited from the laboratory staff of the First Department of Internal Medicine, Niigata University School of Medicine. Each volunteer was informed about the purpose and the procedure of the study before giving written consent to participate. Both studies were approved by the Human Ethics Committee of the respective university. All subjects, including smokers, were normotensive, took no medication, and had no evidence of metabolic or cardiovascular disease. In addition, fresh venous blood was taken from mongrel dogs (weighing from 5 to 28 kg of both sexes, n = 7) in the animal laboratory of Kanazawa Medical University. Animals were handled in a humane way according to the "Guiding Principles for the Care and Use of Laboratory Animals" approved by The Japanese Pharmacological Society.

Hyperventilation study. After the human volunteers were allowed a period of bed rest, control samples of venous and arterial blood were withdrawn from the cephalic vein and brachial or radial artery, respectively. The volunteers were then asked to take deep breaths at 30/min over a period of 5 min using a metronome (3). Immediately after this period of hyperventilation, blood sampling was performed again. Blood gases and hematocrit were analyzed immediately by a blood gas analyzer (ABL625, Radiometer; Copenhagen, Denmark), and a portion of the blood was transferred to 9 vol of hypotonic solution (Tris 10 mM, pH 7.4) to prepare the hemolysate. After vortexing was performed, the hemolysate was centrifuged at 10,000 g for 10 min, and the supernatant was collected and kept at 80°C. Plasma was obtained after centrifugation of the blood at 1,600 g for 5 min at 4°C and was mixed with methanol (1:1) followed by centrifugation at 10,000 g for 10 min at 4°C to remove proteins. The supernatant was collected and stored at 80°C until the analytic procedure.

Measurement of NOx. Determination of plasma NOx was performed by a high-performance liquid chromatography-Griess system (Eicom; Kyoto, Japan) consisting of a separation column, a reduction column (to reduce NO to NO), a flow reactor (with Griess reagent), and a detector at 540 nm as described previously (7). The sensitivity of the setup was 0.1 µM for both NO and NO with a loading volume of 10 µl. When NO and NO in the hemolysate were determined, a C₁₈ reverse phase column (CA-ODS, Eicom) was placed before the separation column to trap hemoglobin, which does not only interfere with the peak of NO, but also affects the baseline of the spectrogram, leading to inaccurate quantification (Fig. 1). Hemoglobin trapping also prevents a rapid fall in the reducting ability of the reduction column that would also contribute to inaccurate quantification. Loading volume was increased to 25 µl to compensate for reduced accuracy by dilution of blood (1/10, see Hyperventilation study). Under these specifications, the sensitivity and detection limit were 0.04 µM. NOx concentrations in whole blood and in RBC were calculated as follows

(1)

(2)

where [NOx] is concentration of NOx and Hct is hematocrit (measured in %). Because very low concentrations of NO and NO were detected in the Tris buffer used to prepare hemolysate, these values were subtracted form those measured in the hemolysate (Eq. 1). This calculation, however, resulted in a small negative value of NO being considered to arise from conversion of preexisting NO in the Tris buffer to NO by hemoglobin. Therefore, NOx value in RBC was expressed as NOx (sum of NO and NO). To minimize NOx contamination, all laboratory ware was washed five times with pure water (resistance > 18.3 M and almost NOx-free through MILLI-Q SP, Millipore; Bedford, MA) (6).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Representative spectrograms of NO and NO in human hemolysate. Hemolysate (25 µl) was loaded on a high-performance liquid chromatography-Griess system (ENO-10) with (B) or without (A) a C18 reverse phase column to trap hemoglobin. A: breakthrough of hemoglobin (Hb) overlaps with the peak of NO. Baseline remains elevated when hemolysate is directly loaded on the system, being a cause of inaccurate quantification of NO and NO in the hemolysate. B: when a C18 reverse phase column is set to trap Hb, clear peaks of NO and NO are obtained. Absorbance is indicated in the ordinate as changes in millivolts (1 mV corresponds to 0.001 absorbance unit) of detector. Abscissa represents the retention time in minutes.

Statistical analysis. All data were expressed as means ± SE. Differences between groups were examined for statistical significance by paired or unpaired t-test where appropriate. Differences were considered to be significant at P < 0.05. Correlations between two variables were evaluated by least-squares regression analysis, and significance was accepted at P < 0.05.

	RESULTS

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Verification of the method. Recovery experiment (ex vivo) was performed using fresh venous blood samples obtained from five volunteers. NOx concentration in the hemolysate was 25.4 ± 6.3 µM in the control state. Addition of 3 µl of NaNO solution (10 mM) to 1 ml of whole blood (expecting a resultant increase of 30 µM of NO in whole blood) followed by gentle agitation at room temperature resulted in an increase of NOx concentration to 54.3 ± 6.0 µM in the hemolysate. The increase was 28.7 ± 1.8 µM, and the recovery ratio was 96.5 ± 4.6%. The increases in NOx in both plasma and RBC were parallel and stable for 60 min (Fig. 2). Standard (mixed) plasma and hemolysate were prepared from blood samples of three volunteers and served to verify the quantification system. Areas under NO and NO curves of the standard hemolysate relative to those of standard solutions (10 µM each) in the absence of ODS column (1.28 ± 0.12% and 75.43 ± 0.29%, respectively, n = 6) were significantly (P < 0.01) smaller than those in the presence of ODS column (2.02 ± 0.08% and 77.11 ± 0.25%, respectively, n = 6). The means ± SE and intra-assay coefficient of variance (in parentheses) of NOx in the standard (n = 6) were 36.30 ± 0.04 µM (0.26%) for plasma, 30.32 ± 0.11 µM (0.80%) for hemolysate, and 22.99 ± 0.23 µM (2.25%) for calculated RBC NOx concentration. Values for interassay (determined 6 days later) were 35.97 ± 0.15 µM (0.95%) for plasma, 30.78 ± 0.28 µM (2.07%) for hemolysate, and 24.41 ± 0.55 µM (5.03%) for calculated RBC NOx concentration. Venous blood freshly drawn from seven mongrel dogs was also subjected to the evaluation. Mean NOx concentration of RBC (10.03 ± 2.06 µM) was significantly (P < 0.05) lower than that of plasma (21.38 ± 5.18 µM), and the resultant NOx ratio (RBC NOx concentration/plasma NOx concentration) was 0.50 ± 0.04 (0.41-0.63 in range).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Distribution of exogenously added NO in whole blood. Solution of NaNO3 (10 mM, 3 µl) was added to 1 ml of whole blood, and changes in NOx concentration in the plasma and red blood cells (RBC) were examined. Recovery of NOx in hemolysate was 96.5 ± 4.6% after 2 min and 95.5 ± 6.0% after 60 min of the addition. When added NOx is expected to distribute only into plasma (hematocrit of 42.6 ± 1.8%), plasma NOx should increase up to 99.8 ± 6.0 µM. However, it is evident that NO distributes to plasma and RBC at a constant ratio, being precisely determined by our system.

Hyperventilation study. As shown in Table 1, arterial PCO₂ and HCO significantly decreased, whereas PO₂ and pH increased after 5 min of hyperventilation, indicative of respiratory alkalosis. A similar result was also obtained in venous blood. However, no significant change by hyperventilation was observed in arterial and venous plasma NOx concentration, or in the RBC NOx concentration (Table 1). Furthermore, the NOx ratio remained stable at around 0.5 (Table 1). Linear regression analysis showed a significant correlation between plasma NOx concentration and whole blood NOx concentration or RBC NOx concentration (Fig. 3, A and B). However, there was no significant correlation between PCO₂ and the natural logarithm of NOx ratio [ln (NOx ratio)] and between plasma HCO concentration and natural logarithm of NOx ratio as is shown in Fig. 3, C and D.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   A: relationship between plasma NOx concentration and whole blood NOx concentration calculated from NOx concentration of hemolysate and Tris buffer (Eq. 1 in MATERIALS AND METHODS). B: relationship between plasma NOx concentration and NOx concentration in red blood cells (RBC NOx) calculated from whole blood NOx concentration and hematocrit (Eq. 2 in MATERIALS AND METHODS). C: relationship between PCO2 and the natural logarithm of NOx ratio (RBC NOx concentration/plasma NOx concentration). D: relationship between plasma HCO concentration and the natural logarithm of NOx ratio.

	DISCUSSION

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Using the high-performance liquid chromatography-Griess system with a trap for hemoglobin, we were able to determine with high accuracy and reproducibility the concentrations of NOx in whole blood samples as well as in plasma and then calculate RBC NOx concentration. The NOx ratio (RBC/plasma) was around 0.5-0.7 and exogenously added NO to whole blood rapidly redistributed to both compartments according to the ratio. The rapid movement of NO across the RBC membrane has been suggested to be due to the Cl/NO exchange system in erythrocytes (4, 12). However, in our additional experiments, pretreatment of human venous blood with 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acidsodium salt (300 µM; n = 6), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid disodiumsalt, 100 µM; n = 4), or dipyridamole (50 µM; n = 4) at high enough concentrations to inhibit anion-exchanger in RBC (10) did not affect NOx distribution before and after exogenous addition of NO (30 µM in final concentration in whole blood in the same situation as is shown in Fig. 2; data not shown). Therefore, the Cl/NO exchange system might not be responsible for the NOx movement in our experimental situation (venous blood, ex vivo at room temperature).

Hyperventilation induced wide changes in PCO₂ and HCO concentration. However, these changes did not influence the NOx ratio (and its natural logarithm) between plasma and RBC, although it is possible that hyperventilation may evoke some compensatory mechanisms for regulation by PCO₂ andHCO. Our results also showed that NOx concentration in RBC was lower than in plasma. Our results are inconsistent with those reported in the study of Recchia et al. (12), who measured NOx concentration in RBC of conscious dogs. The first difference is the NOx ratio, which was markedly higher (4.38-14.6) than that in our present study with canine venous blood (0.41-0.63) and with human arterial and venous blood (0.5-0.7). These values should reconcile with the apparent volume of distribution (V_d) of NOx (nearly equal to V_d of NO). The reported volume of distribution in dogs is 21.5% of body weight (16). Roughly estimated volumes of plasma and RBC are 4.4 and 3.6% of body weight, respectively, assuming an Hct of 45%. Because the V_d has been determined by NOx concentration in venous blood (16), the mean NOx ratio calculated in peripheral venous blood based on the reported values (6.76, n = 6) (12) should be applied. A simple calculation that 4.4% (apparent V_d in plasma) + 6.76 × 3.6% (apparent V_d in RBC) = 28.7% (apparent V_d within only circulating blood), in excess of V_d (21.5%), indicates that the NOx concentration in RBC reported in their study may be too high, whereas our value obtained by canine venous blood (0.5) is fairly reasonable by the same estimation. Similarly, the calculated NOx ratio in our human study indicates that the V_d within circulating blood (8% of body weight) is 6.7% of body weight, being acceptable when the V_d of 28-33% of body weight in humans (1, 9, 13, 15) is taken into consideration.

A possible cause of the high NOx concentration in whole blood in the study of Recchia et al. (12) would be the use of an ultrafiltration unit to remove hemoglobin, because we could not recognize species difference between dog and human in this study. As we have reported previously (6), ultrafiltration units (especially filters) are heavily and variably contaminated with NO and to a lesser degree with NO. Indeed, significant amounts of NO (13 ± 1 pmol; range, 10-15 pmol, n = 8) and NO (400 ± 26 pmol; range, 332-557 pmol, n = 8) in the filtrate (around 150 µl) through a 50-kDa cutoff filter (Millipore) for removal of hemoglobin were noticed in our preliminary studies (phosphate buffer was used as a substitute for hemolysate). The above contamination would be sufficient to result in an erroneously high concentration of NOx in the filtrated hemolysate, because the smaller volume of the hemolysate filtrate (about one-third compared with phosphate buffer in our preliminary studies) would result in higher concentration of NOx in the filtrate and the above calculation (multiplication by dilution factor) would magnify the contamination and error.

Because the RBC NOx values reported by Recchia et al. (12) are difficult to accept, it would not be surprising that close relationships between the natural logarithm of the NOx ratio (their index of the ratio of NOx distribution between plasma and RBC) and PCO₂ or plasma NO concentration (12) are not recognized in our study. Instead, a close relationship between plasma NOx and hemolysate NOx and a rather rough but significant relationship between plasma NOx and RBC NOx were recognized. However, our results do not necessarily exclude the possible redistribution of NOx between plasma and RBC. As shown in Fig. 3B, there are some data points that are far away from the regression line between plasma NOx and RBC NOx concentrations. In addition, our preliminary study showed that the NOx ratio sometimes varied in the same subject under certain conditions (data not shown). Therefore, certain factor(s), other than PCO₂ and HCO, may be operative in determining the NOx ratio between plasma and RBC. Further studies are required to determine these factors.