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The Impact of Surgery and Stored Red Blood Cell Transfusions on Nitric Oxide Homeostasis

Nagababu, Enika PhD; Scott, Andrew V. BS; Johnson, Daniel J. BS; Goyal, Aakshit MD; Lipsitz, Joshua A.; Barodka, Viachaslau M. MD; Berkowitz, Dan E. MD; Frank, Steven M. MD

doi: 10.1213/ANE.0000000000001392
Cardiovascular Anesthesiology: Original Clinical Research Report

BACKGROUND: Cell-free hemoglobin (Hb) forms in stored red blood cells (RBCs) as a result of hemolysis. Studies suggest that this cell-free Hb may decrease nitric oxide (NO) bioavailability, potentially leading to endothelial dysfunction, vascular injury, and multiorgan dysfunction after transfusion. We tested the hypothesis that moderate doses of stored RBC transfusions increase cell-free Hb and decrease NO availability in postoperative surgical patients.

METHODS: Twenty-six patients undergoing multilevel spine fusion surgery were studied. We compared those who received no stored RBCs (n = 9) with those who received moderate amounts (6.1 ± 3.0 units) of stored RBCs over 3 perioperative days (n = 17). Percent hemolysis (cell-free Hb), RBC-NO (heme-NO), and plasma nitrite and nitrate were measured in samples from the stored RBC bags and from patients’ blood, before and after surgery.

RESULTS: Posttransfusion hemolysis was increased approximately 3.5-fold over preoperative levels (P = 0.0002) in blood samples collected immediately after surgery but not on postoperative days 1 to 3. Decreases in both heme-NO (by approximately 50%) and plasma nitrite (by approximately 40%) occurred postoperatively, both in nontransfused patients (P = 0.036 and P = 0.026, respectively) and transfused patients (P = 0.0068 and P = 0.003, respectively) and returned to preoperative baseline levels by postoperative day 2 or 3. Postoperative plasma nitrite and nitrate were decreased significantly in both groups, and this change was slower to return to baseline in the transfused patients, suggesting that blood loss and hemodilution from crystalloid administration contribute to this finding.

CONCLUSIONS: The decrease in NO metabolites occurred irrespective of stored RBC transfusions, suggesting this decrease may be related to blood loss during surgery and hemodilution rather than to scavenging of NO or inhibition of NO synthesis by stored RBC transfusions.

From the Departments of *Anesthesiology/Critical Care Medicine and Biomedical Engineering, The Johns Hopkins Medical Institutions, Baltimore, Maryland; and Department of Radiology, Era Medical College, Lucknow, India.

Accepted for publication March 31, 2016.

Funding: Support was provided from institutional and/or departmental sources as well as Haemonetics Corp. (Braintree, MA) (to SMF) and the New York Community Trust (New York, NY) (to SMF). Support was also provided by the National Institutes of Health (Bethesda, MD) R01 HL105269 (to DEB).

Conflict of Interest: See Disclosures at the end of the article.

Reprints will not be available from the authors.

Address correspondence to Steven M. Frank, MD, Department of Anesthesiology, Johns Hopkins Hospital, Sheik Zayed Tower 6208, 1800 Orleans St, Baltimore, MD 21287. Address e-mail to sfrank3@jhmi.edu.

Physicians transfuse stored red blood cells (RBCs) to compensate for blood loss caused by trauma or surgery and to treat severe anemia in hospitalized patients. Although blood transfusion can be life saving, some concerns remain regarding transfusion-associated morbidity and mortality.1,2 RBC transfusion frequently is reported to be associated with immune suppression, increased infection, increased inflammation, and cardiovascular dysfunction.3–5 In particular, vascular injury and vascular dysfunction may contribute to adverse effects of stored RBCs transfusion.6 Several studies suggest that the biochemical and morphological changes that occur during the storage of RBCs, collectively referred to as “storage lesion,” primarily are responsible for these adverse effects.7 Despite these known adverse changes in stored blood, this field of study is controversial because there are 4 recent randomized clinical trials that have shown no difference in outcomes comparing fresher with older blood.8–11 On the basis of these recent negative studies, it is possible that the storage lesion has no clinically significant impact; however, the trials included few blood units near the end of their 6 week storage duration limit.

Several studies have shown that formation of cell-free hemoglobin (Hb) as a result of hemolysis and Hb-containing microvesicles increases in stored RBCs with increasing the storage duration.12,13 Free Hb and Hb present in microvesicles oxidize vascular nitric oxide (NO) much faster than RBC-encapsulated Hb to form nitrate.14,15 Presence of any cell-free Hb limits NO diffusion from endothelium to smooth muscle cells for activation of guanylyl cyclase; as a consequence, mean arterial blood pressure increases.12,15 Several studies in animals have supported the idea that infusion of free Hb, stored RBC supernatant (preservation solution + plasma), and Hb-containing microvesicles causes vasoconstriction, vascular dysfunction, and vascular injury.12,16–18 In humans, infusion of RBCs stored for longer durations has been shown to significantly reduce brachial artery flow-mediated dilation12 and acetylcholine-stimulated forearm blood flow compared with fresher blood.19,20 In addition to cell-free Hb, membrane structural modification, externalization of phosphatidylserine,21 decreased cell membrane deformability,22,23 and increased endothelial adherence24 could alter vascular NO homeostasis.

To the best of our knowledge, no studies have been conducted to assess the extent of hemolysis and fate of NO metabolism in surgical patients after transfusion of stored RBCs. Hence, we investigated the effect of stored RBC transfusion on vascular NO metabolism by measuring posttransfusion hemolysis, RBC-NO (heme-NO), and plasma nitrite alone and nitrite and nitrate (NOx) concentrations in patients undergoing surgical procedures and receiving standard-issue stored RBC transfusions at our institution. In the current observational study in patients undergoing multilevel spinal fusion surgery, we tested the hypothesis that moderate doses of stored RBC transfusions increase intravascular cell-free Hb and decrease NO availability in surgical patients.

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METHODS

After receiving approval from the IRB at the Johns Hopkins Hospital and obtaining written informed patient consent, we studied 33 patients who were undergoing multilevel spine fusion surgery. Inclusion criteria were age ≥18 years and willingness to receive allogeneic transfused blood components and consent to participate in the study. The only exclusion criteria were any condition affecting RBC morphology (eg, sickle cell anemia, thalassemia). Two cohorts of patients were analyzed: (1) those requiring no stored RBCs (n = 9), and (2) those requiring moderate dose (4–12 units) stored RBC transfusion (n = 17). Patients requiring 1 to 3 stored RBC units (n = 7) were not included because we wanted clear separation between groups, and the adverse effects of stored blood transfusion have been shown to be associated with larger number of units (≥4 units).12 No a priori power analysis was performed because the patients reported in the current study were originally enrolled as part of another clinical study, and these results were a secondary outcome. All patients received general anesthesia with a combination of propofol, isoflurane, fentanyl, and vecuronium. Isotonic crystalloid (either normal saline or lactated Ringer’s solution) was administered according to routine practice. During surgery, an autologous blood salvage system was used (Cell Saver Elite, Haemonetics Corp, Braintree, MA) for processing of shed blood, and these RBCs were transfused preferentially. When salvaged RBCs were insufficient, allogeneic stored RBCs were given. The intraoperative Hb transfusion trigger was 9 g/dL, and the postoperative trigger was 8 g/dL.

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Blood Collection and Processing

Venous blood samples were collected into 2 different individual heparinized tubes preoperatively (Preop), upon completion of surgery (Postop), and on postoperative day (POD) 1, POD2, and POD3. Proper precautions were taken to avoid artificial hemolysis while collecting blood and processing of samples (avoiding small-gauge needles, vigorous suction, agitation, and prolonged tourniquet times). Blood samples used to determine heme-NO, nitrate, and nitrite levels were centrifuged immediately after collection at 1397 g for 3 minutes in a portable centrifuge at the bedside. The centrifuged sample was kept on ice and then transported to the laboratory. Plasma was separated and stored at −85°C immediately and heme-NO in RBCs was measured immediately by a chemiluminescence method (see Heme-NO in RBCs section).

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Stored RBCs

Leukocyte-reduced concentrated RBC units stored in AS1 preservation solution were used for transfusion. No patient received washed RBC units. Samples of RBCs for analysis were drawn from intraoperatively transfused RBC unit blood bags and processed similarly to the patient blood samples described previously. RBC samples from postoperatively transfused units were not collected for testing.

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Whole Blood Hb, Hematocrit, and Percent Hemolysis

The Hb spectrum was recorded from 490 to 640 nm in a Lambda 6 spectrophotometer (PerkinElmer, Waltham, MA). We determined the concentrations of Hb and methemoglobin by a least square-fitting program using spectra of oxygenated Hb and methemoglobin at known concentrations (PerkinElmer Quant C v 4.51).25 Hematocrit (Hct) levels in patient blood and stored RBCs were determined by conventional capillary tube centrifugation method. Cell-free Hb in patients’ plasma and in the supernatant of stored RBCs was measured by the direct spectrophotometric Harboe method.26 Percent hemolysis was calculated based on total Hb and Hct values.27

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Heme-NO in RBCs

Circulating RBCs (but not stored RBCs) contain a significant amount of NO associated with heme-iron of Hb.28 Therefore, in the samples drawn from the patients (but not the stored blood bags), we determined this heme-NO by a chemiluminescence method (The Sievers Nitric Oxide Analyzer, model 280i, GE Analytical Instruments, Boulder, CO) by using ferricyanide and sulfanilamide dissolved in glacial acetic acid. The ferricyanide oxidizes Hb, facilitating the release of NO bound to ferrous heme, and sulfanilamide removes free nitrite. This method is used to identify a pool of NO that is associated with the ferric and ferrous heme moiety of Hb without affecting thiol-associated NO species, and we have described these methods in detail previously.28

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Plasma Nitrite

We determined plasma nitrite by the chemiluminescence method by using iodine and iodide29 or ascorbate30 reagents in glacial acetic acid.

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Plasma Nitrite and Nitrate

Plasma nitrate was converted enzymatically to nitrite, and then total nitrite concentration was determined. Briefly, 100 μL of plasma was incubated with 3 μL of 10 mM NADPH, 3 μL of 1 mM FAD, and 3 μL of 10 U nitrate reductase for 1 hour at room temperature. Then nitrite concentration was determined by the chemiluminescence method as described previously.

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Adjustment of Postoperative Plasma NOx for Loss Through Plasma and Hemodilution

Table 1

Table 1

The plasma loss was calculated by subtracting postoperative plasma volume from preoperative plasma volume. The loss of NOx in this plasma was calculated based on the baseline preoperative plasma NOx values (Table 1). The hemodilution was calculated by subtracting postoperative plasma volume from preoperative plasma volume and then added plasma loss. The postoperative plasma NOx values were corrected to this hemodilution. The final postoperative NOx values were adjusted to loss of NOx through plasma and hemodilution (Table 1, more details).

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Statistical Analysis

Differences in the mean measured values within groups and between groups were analyzed by 2-way repeated-measures analysis of variance. Differences in mean measured values over time within only 1 group were analyzed by 1-way analysis of variance. Individual means were compared with an unpaired Student t test. The Tukey-Kramer post hoc test was used to adjust for multiple comparisons of means. Correlation coefficients between continuous parameters were calculated by linear regression analysis. All analyses were performed with Origin 6.1 software (OriginLab Corporation, Northampton, MA). Values are given as mean ± SD, and P < 0.05 defined significance.

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RESULTS

Patient characteristics and transfusion requirements are summarized in Table 2. Age, sex, and the volume of intraoperative crystalloid and colloid solution were not significantly different between the groups. Transfused patients lost significantly more blood than nontransfused patients. Hb and Hct values were significantly lower in transfused patients than nontransfused patients. Patients receiving stored RBCs received an average of 6.1 ± 3.0 units (approximately 50% of total blood volume). Of these, 3.8 ± 3.5 units were infused during surgery, 1.0 ± 1.1 units were transfused in the first POD, 0.8 ± 1.1 units were infused between POD1 and POD2, and 0.6 ± 0.8 units were infused between POD2 and POD3. The volume of autologous salvaged blood returned to the patients was small (<1 RBC unit) and not significantly different between groups (P = 0.015). This small volume was due to inefficient use of the device, with much of the shed blood ending up on sponges, or in another suction container.

Table 2

Table 2

Table 3

Table 3

Table 3 shows the storage duration of RBC units that were administered to 17 patients from the intraoperative period through POD3. In total, 104 stored RBC units were transfused. Of these, approximately 13% were <14 days old, 32% were between 15 and 21 days old, and 55% were >21 days old. The storage duration of the RBCs ranged from 3 to 38 days with an average of 24 ± 7 days. The mean storage duration of RBCs transfused during surgery and on POD1 was 22 ± 6 days, and the average storage duration of RBCs transfused on POD2 and POD3 was 26 ± 7 and 27 ± 7 days, respectively.

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Hemolysis in Stored RBCs and Patient Blood

Figure 1

Figure 1

We determined the cell-free Hb in the supernatant of stored RBCs, as well as in patient plasma, and expressed it as percent hemolysis, which we calculated based on the total Hb and Hct of the sample. The mean percent hemolysis was 0.21 ± 0.13 in stored RBC units and 0.031 ± 0.02 in Preop patient blood (P < 0.0001; Figure 1A). The stored RBCs had approximately 7 times the hemolysis of fresh blood. The mean percent hemolysis of the salvaged blood was 0.41% ± 0.21%. As expected, hemolysis was not significantly different between preoperative samples and any of the postoperative samples (P > 0.30 for all comparisons), for the nontransfused patients in spite of receiving salvaged RBCs (Figure 1B). For stored RBC transfused patients, hemolysis was 3.5-, 1.6-, 1.5-, and 1.3-fold higher in Postop, POD1, POD2, and POD3 samples, respectively, compared with Preop levels. The increase in hemolysis was significantly different from the Preop baseline value at the Postop time point (P = 0.0002). The Postop hemolysis of transfused patients was also significantly greater (P = 0.001) compared with Postop hemolysis of nontransfused patients. The relative volume of salvaged RBCs administered was 5.5% that of stored RBCs, and percent hemolysis in salvaged RBCs was almost double that of stored RBCs. Therefore, salvaged RBCs could contribute to about 10% hemolysis in the Postop samples. No other difference was found between or within groups at any other time period (Figure 1B).

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Heme-NO Species

Figure 2

Figure 2

The heme-NO in RBCs is unstable and essentially gone by 3 hours after blood collection; thus, heme-NO species were not detectable in stored RBCs. Postop heme-NO species were significantly decreased (P = 0.036) in the nontransfused patients and then returned toward baseline level over the next 3 postoperative days (Figure 2A). Similarly, in moderately transfused patients, heme-NO species were significantly decreased in Postop (P = 0.007) and POD1 (P = 0.04) samples and then returned to Preop level by POD3. In transfused patients, about 50% of native cells were replaced by stored RBCs, which are completely deprived of heme-NO. No significant difference, however, was observed in any of the heme-NO values between nontransfused and transfused patients (P > 0.50 for all comparisons). We plotted plasma nitrite levels in Postop samples by linear regression against their corresponding heme-NO values and found that heme-NO species were significantly correlated (R = 0.525, P < 0.0001) with plasma nitrite, supporting previous reports28,31–33 that heme-NO species are generated by reaction of nitrite with deoxyHb (Figure 2B).

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Nitrite in the Supernatant of Stored RBC Units and Patient Plasma

Figure 3

Figure 3

The mean content of nitrite in the supernatant of stored RBCs was 16 ± 5 nM (range, 10–25 nM). For nontransfused patients, plasma nitrite was decreased by 35%, 42%, 21%, and 13% in Postop, POD1, POD2, and POD3 samples, respectively, compared with that at Preop baseline (Figure 3). Plasma nitrite was similarly decreased by 41%, 46%, 36%, and 23% from baseline in the respective samples from transfused patients. Within-group differences from baseline were statistically significant in both groups at the Postop (P = 0.003) and POD1 (P = 0.01) time points, and on POD2 (P = 0.01) in the moderately transfused group. Plasma nitrite levels did not differ significantly between nontransfused and transfused patients at any time point (P > 0.50 for all comparisons).

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NOx Levels in Supernatant of Stored RBC Units and Patient Plasma

Figure 4

Figure 4

The mean NOx species content in the supernatant of stored RBCs was 4.7 ± 1.2 μM, a value 3.8 times less than that of Preop samples (P < 0.0001; Figure 4A). In the nontransfused patients, NOx was decreased by 24%, 28%, 9%, and 7% in Postop, POD1, POD2, and POD3 samples, respectively, compared Preop baseline levels (Figure 4B) with a return to baseline levels on POD2. In the transfused patients, NOx concentrations decreased by 37%, 30%, 23%, and 19% at Postop, POD1, POD2, and POD3, respectively, and remained below baseline levels even on POD3.

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Adjustment of Plasma NOx Levels Based on Blood Loss and Hemodilution in Nontransfused Patients

Nitrate comprises 99% of NOx levels and has a half-life in plasma of 5 to 6 hours.34 Therefore, it is unlikely that circulating nitrate would be reduced by 24% immediately after surgery based on inhibition of NO synthesis alone (Figure 4).35 Thus, we hypothesized that loss of blood during surgery and hemodilution from crystalloid administration might contribute to the observed decrease in plasma NOx levels. We calculated the loss of blood and extent of hemodilution during and after surgery based on the Hct levels for nontransfused patients (Table 1). Hct was significantly decreased (by approximately 20%, P = 0.0087) and hemodilution was significantly increased (P = 0.007) in these patients. Most of the Hct decrease occurred during surgery followed by additional decreases on POD1 through POD3. After accounting for the loss of NOx species through plasma and hemodilution, we found that the plasma NOx levels in Postop and POD1 samples were not significantly different from Preop values (P > 0.30) (Table 1). Therefore, the decrease in NOx at these time points was likely not due to a decrease in NO synthesis but rather loss of blood and increase in plasma volume (hemodilution). After adjusting the plasma nitrate as described previously, we found that the NOx levels were greater on POD2 and POD3, possibly because of NO produced by endothelial nitric oxide synthase (eNOS) or provided in the diet after the Postop and POD1 time points (Table 1). We were unable to calculate the loss of NOx species through shed blood in transfused patients because native blood is mixed with transfused stored RBCs.

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DISCUSSION

The main objective of this study was to test the hypothesis that cell-free Hb formed as a result of hemolysis in stored RBCs decreases NO availability in transfused patients undergoing major surgery. Our findings show that posttransfusion cell-free Hb does transiently increase, but the findings do not support our hypothesis because NO metabolites, measured to assess the NO availability, changed with almost the same pattern in the presence and in the absence of stored RBC transfusions. Thus, it was apparent that blood loss and hemodilution from IV fluids rather than stored RBC transfusion likely played a role in the transient decrease of these NO metabolites.

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Pre- and Posttransfusion Hemolysis

There was no difference in hemolysis between preoperative and postoperative samples in nontransfused patients, suggesting that anesthesia, surgery, and crystalloid administration do not cause RBC hemolysis. Any increase in hemolysis after transfusion of stored RBCs can be attributed to lysis of RBCs during storage or after transfusion (Figure 1B). Others have reported a significant amount of hemolysis and subsequent effect on NO metabolism and vascular dysfunction in animal models after transfusion of RBCs stored for longer durations.12,18,36 An increase in plasma cell-free Hb and vascular dysfunction also has been shown after transfusion of autologous RBCs with long storage duration to healthy human subjects.19,20,37 However, the extent of hemolysis after transfusion of standard-issue stored RBCs in surgical patients has not been well established. Our study establishes that plasma cell-free Hb increases only transiently after transfusion of (an average of) 6 units of leuko-reduced RBCs stored for an average of 24 days in adsol preservative solution in patients undergoing major spine surgery. Our average storage duration was similar to that of RBCs used in 2 recently published multicenter randomized clinical trials (22 and 28 days).10,11

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Plasma Nitrite in Nontransfused and Transfused Patients

Plasma nitrite levels have been shown to be a better marker for the acute changes of NO synthesis in endothelial cells. Our finding that plasma nitrite decreased postoperatively with a slow return to preoperative levels in both nontransfused and transfused patients suggests that stored RBCs, microvesicles, and cell-free Hb do not have a role in decreasing nitrite levels during surgery. It is unlikely that loss of nitrite through surgically shed blood and hemodilution would produce a decrease of 50% from baseline because continuous synthesis of NO by eNOS should replenish the loss of nitrite.

Three possible mechanisms could contribute to the transient decrease in nitrite that we observed. The first is inhibition of NO synthesis by anesthetic agents. In vitro, direct exposure of vascular strips,38,39 brain tissue,40 polymorphoneutrophils,41 and thoracic aorta42 to volatile anesthetic agents such as enflurane, isoflurane, and halothane has been shown to reversibly inhibit NOS activity. However, this inhibition of eNOS was not substantiated in in vivo studies. Isoflurane was used in our study patients, but we do not think it plays a major role in inhibition of eNOS activity. Second, a few studies have shown that surgery or trauma decreases plasma NOx species by decreasing NOS precursors such as arginine, citrulline, and ornithine.43–45 These authors did not consider NOx species and NOS substrate loss through shed blood and hemodilution. Instead, the authors suggested that the arginase released from RBCs as a result of hemolysis caused reductions in arginine levels.46 We cannot exclude the role of arginase, but in our study, plasma nitrite levels were decreased in nontransfused patients, suggesting that RBC arginase may not have a role in decreasing nitrite levels. Third, during surgery, patients were intratracheally intubated, which prevented swallowing of saliva, which may have interrupted the nitrate-to-nitrite pathway, thus contributing to the observed decrease in nitrite. In addition, patients usually are treated with antibiotics after surgery to prevent infections. These antibiotics reduce mouth commensal bacteria that in turn reduce the reduction of nitrate to nitrite. Continuous generation of nitrite is essential to maintain levels in plasma because the half-life of nitrite in blood is approximately 30 minutes.34 Hence, any interruption of recycling in the nitrate-to-nitrite pathway in surgical patients might have decreased plasma nitrite because the average surgery duration was 6.5 hours. This pathway is a likely mechanism for decreasing nitrite levels in both nontransfused and transfused patients. If the nitrate–nitrite recycling pathway contributes to plasma nitrite, using plasma nitrite as a marker for acute changes in endothelial NO synthesis should be reevaluated. Several studies have shown that nitrite is reduced to NO by several heme proteins under hypoxic conditions, providing an eNOS-independent source of NO in the vasculature.28,47,48 Hence, the decrease in plasma nitrite in surgical patients may temporarily affect the nitrite-mediated source of NO.

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Plasma Heme-NO Species

Venous RBCs contain substantially more heme-NO25,28 than do arterial RBCs, and it is believed that these species are involved in nitrite-mediated vasodilation.33 The correlation of heme-NO with plasma nitrite (Figure 2B) supports previous studies, which have shown that heme-NO species are formed when nitrite is reduced by deoxyHb. Thus, the decrease in nitrite levels after surgery appears to be responsible for decreasing heme-NO species. Studies have shown over the last decade that RBCs may sense hypoxia and transfer NO bioactivity to the vasculature from S-nitrosohemoglobin and/or reduce nitrite to NO by deoxyHb.33,47,49,50 The mechanism for release of this NO from RBCs is not yet established. The postoperative decrease in heme-NO and the slow return to baseline levels by POD3 for nontransfused and transfused patients may have affected the RBC-mediated hypoxic vasodilation at least temporarily (Figure 2A) in surgical patients.

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Plasma NOx

Plasma NOx levels are used routinely to assess whole-body NO synthesis. The decreased plasma NOx we found in postoperative samples in both nontransfused and transfused patients (Figure 4) implies a reduction in NO synthesis. Unlike nitrite (Figure 3), acute inhibition of NO synthesis by stored RBCs or surgery itself should not be expected to significantly decrease nitrate in the postoperative period because the half-life of nitrate in circulation is 5 to 6 hours.34 The cell-free Hb in stored RBCs should actually increase nitrate by oxidizing NO to nitrate. Plasma nitrate was not correlated with hemolysis (R = 0.045, P = 0.71), suggesting that cell-free Hb did not scavenge additional endothelial NO (data not shown). The alternate explanation for a postoperative decrease in nitrate may be related to blood loss during and after surgery as well as hemodilution. As we expected, after adjusting the nitrate values based on loss of nitrate through plasma and hemodilution, the postoperative concentrations were comparable to preoperative baseline values in nontransfused patients (Table 1). The slower return to baseline NOx levels in transfused patients could be attributed to loss of more nitrate due to loss of more native blood and more hemodilution. Studies have shown that nitrate concentrates in saliva and is reduced to nitrite by mouth commensal bacterial nitrate reductase and then nitrite is reduced to NO under acidic conditions of the stomach.28,51,52 Both nitrite and NO are vasoactive molecules. The slower return to baseline NOx levels in the surgical patients may have decreased NO availability in postoperative patients through the nitrate–nitrite–NO pathway.

Potential limitations in our study include the following. As with most clinical studies, there are multiple factors occurring simultaneously, which may affect the measured outcomes. For example, patients received both salvaged and stored RBC transfusions during surgery, which precludes a clear comparison of the 2 types of RBCs. There was, however, a small volume of salvaged RBCs given compared with stored RBC, for example, and thus we presumed that postoperative changes in RBC properties were primarily related to the stored blood transfusions. The duration of follow-up after transfusion was limited to 3 days; therefore, we were unable to determine that the normalization of NO metabolites and additional changes of NO metabolism occur after this time. We only studied patients undergoing one type of procedure, and we cannot generalize our findings for patients undergoing other types of surgery. Patients having other procedures, such as those requiring cardiopulmonary bypass, may experience more RBC lysis and increased cell-free Hb, and further studies would be needed to make relevant conclusions in other such clinical settings. We did not randomize or control the duration of blood storage for the units that were transfused. Thus, we did not have patients who received RBCs of exclusively shorter or longer storage duration, and we are unable to clearly delineate whether storage duration or the number of units transfused was the primary variable associated with decreased deformability in postoperative patient blood samples. Last, we were unable to sample every unit of transfused blood to obtain measurements. Most units given intraoperatively were sampled and tested, but none of the units transfused postoperatively was tested.

In summary, hemolysis was increased only transiently after transfusion of stored RBCs, implying that cell-free Hb formed during storage and/or intravascularly is efficiently removed from the circulation. The decrease in circulating NO metabolites occurred in patients with and without stored blood transfusions, suggesting this decrease may be related to blood loss during surgery and hemodilution rather than to inhibition of NO synthesis by stored RBCs. We conclude that in our observed patient population, surgery and its associated procedures affect the eNOS independent source of vascular NO availability to a greater extent than do moderate doses of stored RBCs.

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DISCLOSURES

Name: Enika Nagababu, PhD.

Contribution: This author helped design the study, collect the data, analyze the data, and prepare the manuscript.

Conflicts of Interest: Enika Nagababu reported no conflicts of interest.

Name: Andrew V. Scott, BS.

Contribution: This author helped design the study, collect the data, analyze the data, and prepare the manuscript.

Conflicts of Interest: Andrew V. Scott reported no conflicts of interest.

Name: Daniel J. Johnson, BS.

Contribution: This author helped collect the data, analyze the data, and prepare the manuscript.

Conflicts of Interest: Daniel J. Johnson reported no conflicts of interest.

Name: Aakshit Goyal, MBBS.

Contribution: This author helped collect the data.

Conflicts of Interest: Aakshit Goyal reported no conflicts of interest.

Name: Joshua A. Lipsitz.

Contribution: This author helped collect the data.

Conflicts of Interest: Joshua A. Lipsitz reported no conflicts of interest.

Name: Viachaslau M. Barodka, MD.

Contribution: This author helped design the study, collect the data, analyze the data, and prepare the manuscript.

Conflicts of Interest: Viachaslau M. Barodka reported no conflicts of interest.

Name: Dan E. Berkowitz, MD.

Contribution: This author helped design the study and prepare the manuscript.

Conflicts of Interest: Dan E. Berkowitz reported no conflicts of interest.

Name: Steven M. Frank, MD.

Contribution: This author helped design the study, collect the data, analyze the data, and prepare the manuscript.

Conflicts of Interest: Steven M. Frank has consulted for Haemonetics Corp. (Braintree, MA), a company involved with blood salvage equipment.

This manuscript was handled by: Roman Sniecinski, MD.

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ACKNOWLEDGMENTS

The authors thank Claire Levine, MS, ELS (Manager, Editorial Services, Department of Anesthesiology/Critical Care Medicine, Johns Hopkins Medicine, Baltimore, MD), for editorial assistance.

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