INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

MEASUREMENTS OF NITRIC OXIDE (NO) production are often used to explore the physiological and pathophysiological roles of this important biological mediator. Because of the very short half-life of NO, direct measurements in vivo are technically challenging (6, 8), and most of the studies in this area are based on the determination of nitrite/nitrate concentration in blood as an index for NO production (1, 2, 7, 9, 17). The rationale is that almost all of the NO freely diffusing in blood from cells is rapidly oxidized to form nitrite and nitrate anions (NOx) (14, 17). Nitrate represents ~90% of total NOx (17). For practical reasons, NOx are measured in plasma rather than in whole blood, assuming that the ratio between the concentrations of NOx in plasma and in red blood cells is constant. This assumption has never been tested and has been challenged by a recent study from our laboratory (9) in which we measured the arterial-coronary sinus difference of NOx concentration during the development of pacing-induced heart failure. Consistent with previous results (2, 17), we found a net cardiac production of NOx in healthy animals. Failing hearts, however, were characterized by a net NOx uptake. The uptake was obviously apparent because NOx cannot be recycled and would accumulate in cardiac tissue, reaching toxic concentrations. To explain this apparent uptake, we hypothesized that the partition of NOx between red blood cells and plasma is different in arterial compared with venous blood and that this can cause an underestimation of the venous-arterial difference if NOx are measured only in plasma. The aim of the present study was to test the hypothesis that the ratio between NOx concentrations in plasma and in red blood cells is not constant but varies in relation to chemical parameters of the blood.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Blood samples (n = 19) of 7 ml each were withdrawn with heparinized syringes from the aorta, leg veins, and coronary sinus of seven mongrel dogs and immediately stored on ice. pH, PO₂, and PCO₂ were measured with a blood-gas analyzer (Instrument Laboratory), and plasma HCO₃ concentration was calculated using the Henderson-Hesselbach equation. Care was taken to avoid contact of the blood with air. After pH and gas measurements were obtained, the syringes were tightly capped and centrifuged at 1,000 g for 15 min at 0°C to separate plasma from red blood cells. Plasma and red blood cells were then collected in different plastic tubes. The plasma was stored at 20°C. Red blood cells were lysed by vigorously mixing them with distilled water in a ratio of red blood cells to water of 1:3 (vol/vol). Twenty minutes were allowed to obtain complete lysis. In pilot experiments, we found that no pellet of red blood cells was detectable in centrifuged lysates obtained with this procedure. Three milliliters of distilled water used on the day of the experiment were stored at 20°C and utilized as a blank during NOx determination. The lysate was spun overnight at 1,000 g through ultrafilters with a 50-kDa cutoff (Millipore) to remove hemoglobin. The clear ultrafiltrate was then collected in plastic tubes and stored at 20°C. NOx measurements were performed within 2-3 mo after sample storage. In preliminary tests for a previous study (17), we found that sample storage at 20°C does not affect the reproducibility of results when the measurements are performed within 3 mo (unpublished data).

The protocol was approved by the Institutional Animal Care and Use Committee of the New York Medical College and conforms to the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health.

NOx measurements. NOx was measured in plasma and in the ultrafiltrate. The method for NOx analysis was previously described in detail (17). Plasma was incubated with Aspergillus nitrate reductase to reduce nitrate to nitrite and then acidified to pH < 2 under argon gas to convert nitrite to NO. The gaseous NO was injected into a NO chemiluminescence analyzer (Sievers). In the analyzer, NO combined with ozone to generate a luminescence directly proportional to the amount of NO injected, i.e., to the original NOx in the plasma sample.

Statistical analysis. The ratio between NOx concentration in erythrocyte (eNOx) and plasma (pNOx) was calculated. eNOx/pNOx was plotted against all the measured chemical values of the blood, and potential correlations were evaluated by calculating the best fit, based on least-squares regression analysis. Preliminary tests indicated that the natural logarithm of the ratio, rather than the ratio itself, resulted in linear correlations. The regression coefficient (r) of the regression line was calculated, and significance accepted at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The blood samples were withdrawn from different animals and different vascular beds, so it was possible to obtain a wide range of values for the chemical parameters: 7.35 to 7.42 for pH, 17 to 95 mmHg for PO₂, 32.8 to 50.6 mmHg for PCO₂, and 20.6 to 28.3 mM for plasma bicarbonate. Values of NOx concentrations in plasma and in red blood cells are reported in Table 1. On the basis of the hematocrit (Hct) of blood samples, it was possible to calculate the concentration of NOx in whole blood: NOx (total) = (eNOx × Hct) + [pNOx × (1  Hct)]. The average value of total NOx concentration was 22.71 ± 2.58 µM. This value was consistent with the concentration of NOx measured by Sonoda et al. (13) in whole blood. NOx in red blood cells was higher than in plasma, with eNOx/pNOx ranging from 4.38 to 14.60. Significant, linear, and direct correlations were found only between the natural logarithm of eNOx/pNOx and PCO₂, as shown in Fig. 1A, and between the natural logarithm of eNOx/NOx and bicarbonate, as shown in Fig. 1B. PO₂ did not correlate with the natural logarithm of eNOx/pNOx, although it inversely correlated with PCO₂, as shown in Fig. 1C. On the basis of the equations describing the regression lines, it was possible to calculate that eNOx/pNOx changes by 5% for a 1-mmHg difference in PCO₂ and by 15% for a 1 mM difference in bicarbonate concentration.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   The natural logarithm of the ratio between concentrations of nitric oxide metabolites (NOx) in erythrocytes (eNOx) and in plasma (pNOx) is plotted against PCO2 (A) and bicarbonate (HCO3) concentration (B). C: PO2 is plotted against PCO2. n = 19 Samples. The equation describing the regression line is indicated in each panel.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Our study demonstrates that the ratio between the concentrations of NOx in red blood cells and in plasma is not constant but proportional to PCO₂ and bicarbonate concentration in blood. As a consequence, venous blood has a higher eNOx/pNOx because of the higher content in CO₂/bicarbonate. In fact, although PO₂ did not correlate with eNOx/pNOx, it inversely correlated with PCO₂, indicating that more desaturated blood samples were obviously richer in CO₂. No correlation was found between eNOx/pNOx and pH, yet this parameter plays an important role in determining the concentration of bicarbonate in blood. Our results provide the first evidence of variable NOx redistribution between red blood cells and plasma. Because the correlations were obtained by using the natural logarithm of eNOx/pNOx, small differences in PCO₂ and/or bicarbonate are associated with dramatic differences in the NOx redistribution between plasma and red blood cells. eNOx was indeed 4.4-14.6 times higher than pNOx. In particular, eNOx/pNOx is three times more sensitive to changes in the bicarbonate concentration (determined by PCO₂ and pH) than to changes in PCO₂. We did not identify the mechanisms underlying such a phenomenon; however, this can be explained on the basis of recent and classic studies on the processes of NO oxidation in blood and the mechanisms of ion exchange through the erythrocyte membrane. In plasma, the free radical NO is converted to nitrite and nitrate anions in a ratio of 5:1, but, in the presence of oxyhemoglobin, NO is almost entirely oxidized to form nitrate (14, 15), the main component of NOx. This process involves hemoglobin and therefore occurs inside the red blood cell (Fig. 2A). Part of the newly formed nitrate leaves the erythrocyte and diffuses in plasma, likely exchanging with other anions. It is known, in fact, that the half-time of nitrate exchange with chloride through the erythrocyte membrane is <10 s at 0°C (4). Because nitrate can rapidly exchange with other anions through the red cell membrane, it is very likely that NOx redistribution between erythrocytes and plasma is strongly influenced by the chloride shift occurring in venous blood. As shown in Fig. 2B, CO₂ diffusing from tissues is converted to bicarbonate in red blood cells, and then part of bicarbonate diffuses in plasma exchanging with chloride (10). An opposite ion flow occurs in erythrocytes of arterial blood. It is possible that NOx exchange with chloride and/or bicarbonate during these processes. The importance of NOx in the biophysical equilibrium of the red blood cell can be reasonably considered minimal, because NOx concentration in blood is in the micromolar range, whereas the concentration of bicarbonate, chloride, and other major ions is in the millimolar range. Conversely, this redistribution assumes a major practical importance for studies in which NOx are used as an index of NO synthesis in vivo. To date, a correct estimation of systemic production of NO can be performed only by employing very sophisticated techniques (11). On the other hand, an estimation simply based on NOx concentration in arterial or in venous blood (12, 16) could be misleading, because circulating NOx are characterized by very complex kinetics that depend, for instance, on the volume of distribution and on renal clearance (3, 17). The problems relative to NOx kinetics can be avoided by measuring NO production in single organs as arterial-venous difference of the NOx concentration multiplied by mean blood flow. We, along with other investigators, have adopted this method in several studies (1, 2, 7, 9, 17). However, in light of our present results, this method could be misleading if NOx concentration is measured in plasma rather than in whole blood. For instance, given the very high rate of nitrate ion exchange through the erythrocyte membrane (4), the handling of blood samples could importantly influence the partitioning of NOx between red blood cell and plasma. Plasma should be separated from red blood cells, taking care to avoid exposure of the blood samples to the air, as we did in our study, to prevent possible alterations in blood PCO₂/bicarbonate with consequent changes in NOx redistribution. Even if this precaution is adopted, however, measurements in plasma cause a systematic underestimation of NOx production, because a bigger fraction of total NOx accumulates in erythrocytes of venous compared with arterial blood. We previously found a net production of NOx only by the heart and not by other organs (17). This was somewhat surprising, because it is very unlikely that the heart is the only source of circulating NOx. Our present data suggest that the production of NOx by any organ was previously underestimated. A second implication is that, in the absence of any additional NO produced by a given organ, a higher concentration of NOx in erythrocytes of venous blood could be achieved only after a net transfer of NOx from plasma to red blood cells. The theoretical result would be a positive arterial-venous difference of NOx concentration in plasma across the organ with an apparent uptake of NO metabolites. This was indeed our finding in failing hearts (9), in which the endothelial NO synthesis in microvascular endothelium was reduced (18). Other authors found cardiac NOx uptake in normal and failing hearts (5). To avoid these errors, NOx should be measured in the whole blood. Methods to measure total NO-related compounds in whole blood have been described by Sonoda et al. (13). These authors measured total NO-related compounds, including water-soluble NOx and membrane- and protein-bound NO, in whole blood denatured by thermolysis and then ultrafiltrated.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   A: nitric oxide (NO) is mainly synthesized in endothelial cells and diffuses in the vascular lumen. NO reacts with oxyhemoglobin (HbO2) in red blood cells to form methemoglobin [Hb(Fe3+)] and nitrate (NO3). B: proposed mechanism of NO3 partitioning between plasma and red blood cells. In venous blood, HCO3 leaves the red blood cell by following the concentration gradient, and the electrolytic equilibrium is maintained by a simultaneous entry of Cl (chloride shift). An opposite exchange occurs in arterial blood. Nitrate, which is also an anion, could be involved in these anion exchanges.

Three limitations of our study should be considered. First, as already discussed, we did not explore the cause of different NOx redistribution between arterial and venous blood; however, this was beyond the aim of our study. Second, we measured only water-soluble products of NO oxidation and neglected other NO-related compounds, such as S-nitrosoalbumin and nitrosylhemoglobin; however, they represent a very small portion of total NO derivatives in blood (13, 14). Third, NOx were measured separately in plasma and erythrocytes only to determine the partitioning between the two compartments. This method should not be used to measure total NOx, because calculations based on concentrations determined in plasma and red blood cells could add further variability and result in less precise measurements than direct NOx measurements in whole blood. In two dogs (dogs 1 and 4 in Table 1), indeed, we found one positive arterial-venous difference of NOx concentrations calculated for the whole blood, indicating an unrealistic NOx uptake by tissues.

In conclusion, measurements of plasma NOx may not provide an accurate estimate of total NOx in blood. The margin of error due to the variable redistribution of NOx between plasma and red blood cells can be disregarded only when samples with very close CO₂/bicarbonate concentrations are compared. To avoid a large underestimation of venous-arterial differences in NOx content, NO-related compounds should no longer be measured in plasma but rather in whole blood.