Anesth Analg 2001;92:324-328
© 2001 International Anesthesia Research Society
CARDIOVASCULAR ANESTHESIA
The Effects of Diaspirin Cross-Linked Hemoglobin on the Tone of Human Saphenous Vein
Mark R. Forrest, FRCA,
Adrian H. Chester, PhD*, and
David Royston, FRCA
Departments of Anaesthesia and *Pharmacology, Harefield and Royal Brompton NHS Trust, Harefield, London, UK
Address correspondence and reprint requests to Dr. Mark Forrest, Consultant Anaesthetist, Department of Anaesthesia, Manchester Royal Infirmary, Oxford Road, Manchester, UK, M13 9WL. Address e-mail to mforrest50{at}hotmail.com
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Abstract
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Diaspirin cross-linked hemoglobin (DCL-Hb), when infused into animals, causes vasoconstriction thought to be caused by nitric oxide (NO) binding by the hemoglobin molecule. The purpose of this study was to ascertain whether DCL-Hb causes vasoconstriction in human saphenous vein taken from patients undergoing myocardial revascularization and whether NO scavenging is the mechanism. The direct effect of DCL-Hb on saphenous vein tone was tested by adding increasing concentrations (10-8 to 10-5M) of the drug. In an additional series of experiments, the influence of DCL-Hb on the dilator response to endothelial dependent and independent vasodilators was tested. This was achieved by attempting either to reverse the effects of acetylcholine, sodium nitroprusside, or S-nitrosylglutathione with prior incubation with DCL-Hb or to inhibit the dilator response in vessels preconstricted with 10-6M norepinephrine. There was no effect of DCL-Hb alone on saphenous vein tone. DCL-Hb significantly reduced vasodilatation with all vasodilators (P < 0.05). After maximal relaxation with sodium nitroprusside and s-nitrosylglutathione, there was significant vasoconstriction with DCL-Hb at concentrations larger than 10-6M, (P < 0.05). The authors conclude that DCL-Hb does not constrict human saphenous vein but can affect vessel tone by reversal of the effect of endogenously or exogenously released NO.
Implications: Hemoglobin solutions are used as alternatives to blood transfusion. In animals, they cause vasoconstriction by binding nitric oxide. This is an in vitro study of the effects of one hemoglobin solution in human blood vessels. It shows that this hemoglobin solution alone had no effect in human tissue but antagonized other agents used to alter vessel tone.
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Introduction
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There has been a continuing search for oxygen-carrying solutions to be used as substitutes for allogeneic whole blood. One approach has been the development of hemoglobin-containing solutions that can be manufactured and purified to the same standards as other parenteral fluids. Lamy et al. (1) recently reported the efficacy of diaspirin cross-linked human hemoglobin solution (DCL-Hb; Baxter Healthcare Corp, Deerfield, IL) in reducing the need for transfused allogeneic packed erythrocytes in a European multicenter study of patients having routine myocardial revascularization.
DCL-Hb is a solution of stabilized human hemoglobin with the 
-subunits cross-linked by a covalent bond (2). Infusion of DCL-Hb causes marked increases in both systemic and pulmonary arterial pressures associated with increases in systemic and pulmonary vascular resistance (3–8). This effect might even have a therapeutic benefit by allowing the discontinuation of use of vasopressors in patients in intensive care (9). Conversely, in humans, the pressor effect was without evidence of decreased peripheral perfusion (10). Yet increases in vascular resistance in specific organs, for example, the coronary circulation, could constitute a potential adverse effect. Only limited data are available in human tissues. Infusion of free hemoglobin into human coronary arteries in situ has no effect on vessel diameter under basal conditions but reverses the increased diameter that results from infusion of acetylcholine (ACh) (11).
Nitric oxide (NO) has a high affinity for iron-containing molecules. The iron component of the heme molecule may therefore interfere with the vasodilator action of the NO system, and scavenging of NO by hemoglobin may be the cause of vasoconstriction (5,12,13). In vitro experiments on isolated blood vessels from animal sources have confirmed a pressor effect of DCL-Hb, although the magnitude of the responses varied widely with the species (14,15). This species difference is possibly related to the basal release of NO from endothelium.
Given the species difference and the common, almost universal use of saphenous vein in myocardial revascularization, it would be useful to determine any effect of hemoglobin solutions in this tissue. The aim of this study was to determine the effect of DCL-Hb on resting vascular tone and any potential to inhibit the relaxation of saphenous vein produced by an NO-dependent process.
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Methods
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Saphenous vein was taken from 10 patients undergoing myocardial revascularization with IRB approval. No patients were taking inhibitors of angiotensin-converting enzyme, but all were taking ß-adrenoceptor antagonists and long-acting nitrates, typically isosorbide mononitrate. Vein samples were taken from a subset of patients undergoing myocardial revascularization. There were no specific exclusion criteria, although vein previously distended by the surgeon was not used, and no patient refused to allow use of saphenous vein surplus to requirements for revascularization.
The vein was placed in a modified Tyrodes solution and immediately transported to the laboratory for study. Veins were dissected free of adherent fat and subcutaneous tissue and cut into rings approximately 4 mm wide. The vein was suspended from two intraluminal hooks and bathed in modified Krebs solution (composed of 136.9 mmol/L NaCl, 11.9 mmol/L NaHCO3, 2.7 mmol/L KCl, 0.4 mmol/L NaH2PO4, 2.5 mmol/L MgCl2, 2.5 mmol/L CaCl2, 11.1 mmol/L glucose, and 0.04 mmol/L disodium EDTA) and gassed with 95% oxygen and 5% CO2. The organ baths were maintained at 37°C by a water jacket. One of the hooks was attached to a Grass FTO-3C strain gauge transducer (Grass Electronics, Quincy, MA) connected to a polygraph. An optimal preload was then applied to each segment, and after a period of 40–60 min the vessel rings had relaxed and a steady baseline was achieved. Potassium chloride (KCl) 90mM was then applied to assess tissue viability. After washout of the KCl from the tissue bath, the vessel rings were again allowed to relax to a steady baseline. One segment from each vein was treated with ACh to assess endothelial integrity; dilation of 20% of maximal constriction was taken as evidence of intact endothelium, as previously reported (16,17). After a further period of stabilization, the following three experiments were performed.
First, DCL-Hb or 4.5% human albumin solution (HAS) was applied to the segments in cumulative concentration increments (dose range 10-8 to 3 x 10-5M) in half-log units. Second, after constriction with norepinephrine (NE) 10-6M, either DCL-Hb (10-5M) or HAS 4.5% was applied to the vein rings. Having been allowed half an hour for any response to stabilize, the vein rings were then treated with increasing concentrations of either ACh (10-9 to 10-4M), sodium nitroprusside (SNP) (10-8 to 10-5M), or s-nitrosylglutathione (GSNO) (10-9 to 10-4M). Lastly, after constriction with NE and maximal relaxation with either SNP or GSNO, the vein rings were treated with increasing concentrations of either DCL-Hb (10-8 to 10-5M) or HAS 4.5%.
Data were expressed as a percentage relaxation of the contractile response to NE 10-6M. Mean (SEM) responses were calculated. Statistical analysis comparing differences between treatment groups was performed by using the Kruskall-Wallis test followed by a Mann-Whitney U-test. Differences within the groups were evaluated with analysis of variance followed by a Wilcoxon matched pair test. All statistical analysis was two-tailed at the 0.05 significance level.
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Results
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Thirty-four ring segments were used in this study. Rings that failed to respond to either KCl or ACh were excluded from the study (n = 2). Neither DCL-Hb (10-8 to 3 x 10-5M) nor 4.5% HAS had an effect on the resting tone of segments of saphenous vein.
Dilation of preconstricted ring segments by ACh was concentration dependent and biphasic, with a maximal effect at 10-6M for both control and DCL-Hb treated rings. However, this maximum magnitude of dilation was significantly less with DCL-Hb treatment: 7.63% (1.2) versus the control response of 23.4% (7.2), P = 0.01 ( Fig. 1A). At the maximum concentration of ACh studied (10-4M), the control response was to produce a 9.5% (5.4) relaxation, whereas with DCL-Hb there was a constrictor effect of 5.87% (7.1); P = 0.03 above the baseline value.
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Figure 1. Human saphenous vein rings preconstricted with norepinephrine (10-6M) and the effects of increasing concentrations of vasodilators examined. Acetylcholine (Fig. 1A) has direct constrictor and nitric oxide-dependent dilator actions, which explains the biphasic response. Both sodium nitroprusside (SNP) (Fig. 1B) and s-nitrosylglutathione (GNSO) (Fig. 1C) liberate nitric oxide by direct means and can overcome the nitric oxide binding provided by the diaspirin cross-linked hemoglobin (Hb). Data are expressed as mean ± SEM; * P < 0.05; # P < 0.01 comparing groups at each dose of dilator applied.
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Dilation of the segments to SNP and GSNO increased with increasing dose. For SNP, maximal dilation occurred at 10-5M and was not significantly different at 102% (2.70) for the control group and 91.1% (3.71) for the DCL-Hb group (Fig. 1B). For GSNO, the maximum response was also achieved at 10-5M with relaxation for control and DCL-Hb groups of 87.4% (11.3) and 55.2% (16.8), respectively (P = 0.07) (Fig. 1C). For both the SNP and GSNO experiments, DCL-Hb significantly reduced the relaxation response produced by doses below the maximum.
In the last series of experiments, after maximal relaxation with SNP and GSNO, there was a significant dose-dependent constriction with DCL-Hb at concentrations more than 10-6M ( Fig. 2).
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Figure 2. Effect of increasing concentrations of diaspirin cross-linked hemoglobin (DCL-Hb) on saphenous vein rings maximally relaxed with SNP (sodium nitroprusside) and GSNO (s-nitrosylglutathione). Data are expressed as mean ± SEM. * P < 0.05 compared with the smallest concentration of DCL-Hb (10-8M).
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Discussion
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This study found no de novo alteration in vessel tone when saphenous vein rings were exposed to DCL-Hb at concentrations up to 10-5M. These findings differ from those reported by Muldoon et al. (15) in porcine pulmonary veins treated with cross-linked hemoglobin under basal conditions, and are more in keeping with the lack of effect of hemoglobin on basal tone of human coronary artery (11) and the small effects of hemoglobin solutions observed in rat and dog arterial vessels (14). These differences are most likely related to baseline production of NO. Tissues with more baseline NO, for example, porcine pulmonary artery, exhibit more response to DCL-Hb (14,15). Our observations suggest that baseline expression of NO by human saphenous vein is low, possibly because of absence of flow, which stimulates release of NO in vivo, in our experimental system. Other vessels used as conduits may have larger baseline NO expression (17). Whether these vessels show a direct response to hemoglobin solution is unknown.
Our study demonstrated that, with all three vasodilators tested, when human saphenous vein was initially constricted with NE and then dilated, treatment with DCL-Hb caused reconstriction. The scale of this effect was dependent on the nature and dose of the dilator.
ACh produced a bimodal effect because of its two divergent actions. First, it acts via receptors on the endothelium to increase NO release, and this acts on the surrounding muscle, producing vasodilatation. Larger concentrations of ACh stimulate muscarinic receptors within the smooth muscle, causing contraction. The shape of the dose-response curve reflects the vasoconstrictor effect overcoming the NO-dependent dilation. Our results in the ACh study support data from human coronary artery (11) and the hypothesis that DCL-Hb alters vascular tone by binding NO. The incomplete relaxation in response to ACh suggests that saphenous vein has a low endothelial NO-generating capacity to this agonist, which was fully bound by DCL-Hb. This resulted in a net constrictor effect at larger concentrations of ACh. This result mirrors studies of human coronary artery in vivo, in which infusion of free hemoglobin-constricted vessels previously dilated with ACh (11).
In vessels producing larger quantities of NO than saphenous vein, this may saturate the DCL-Hb, and restoration of normal vascular tone will follow. Resetting of the NO-inhibitor balance may explain observations made in patients after cardiac surgery, of a small statistically significant increase in mean arterial pressure after the first infusion of hemoglobin solution but no increases with further infusions (1).
When exogenous NO was added to the system through release from SNP or GSNO, then the relaxation produced was much more, showing the capacity of the muscle of the vein wall to respond to NO concentrations larger than that produced by its local endothelium. As shown by the divergence between curves (Fig. 1, B and C), DCL-Hb was capable of "mopping up" the NO required to induce submaximal relaxation of the vessel wall. However, the DCL-Hb and control curves converged at larger, maximal relaxation concentrations for SNP and GSNO, suggesting that DCL-Hb had a maximum binding capacity for NO. In the studies reported here, the inhibiting effect of 10-6M DCL-Hb was overcome by both SNP and GSNO at 10-5M concentration. However, increasing the concentration of DCL-Hb reduced the dilator effect of these agents (Fig. 2). We did not investigate the effects of hemoglobin solution on saphenous vein dilated in a non-NO dependent fashion.
Two clinical hemodynamic states can be postulated with either a normal or small NO production and normal to high vascular tone, as may be found after heart surgery, or the opposite, as may be found with sepsis.
In the case of heart surgery, exogenous vasodilators will work in the presence of DCL-Hb, albeit at larger doses. However, because all vasodilators are given to hemodynamic effect, this should have no implications related to patient well being. If a steady-state administration of a vasodilator has been achieved, then the administration of a hemoglobin solution such as DCL-Hb will likely reduce the biological effectiveness of this infusion.
This is not the case with an increased production of NO, as in sepsis. In this condition, the administration of hemoglobin solution will produce a short-lived benefit to improve systemic blood pressure and regional blood flow (1,10,18,19). In vitro studies suggest that part of mechanism of this action is caused by NO binding to iron in hemoglobin. These data are supported by in vivo studies demonstrating modulation of cardiovascular effects of DCL-Hb by the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (20,21), and L-arginine (an NO precursor) (20). This property may be of therapeutic value in conditions with increased endothelial NO expression, notably in septic shock (22). For instance, hemoglobin solutions cause hemodynamic improvement when given to septic sheep (23), and DCL-Hb has been studied as a potential vasopressor in critically ill humans (9).
As with the observations after cardiac surgery (1), these pressor effects were short lived, possibly because of a resetting of the vascular tone. However, and most disappointing, are the preliminary observations in patients with trauma or sepsis, in whom the administration of drugs designed to prevent production of NO (such as false substrate) or bind NO (such as hemoglobin) are associated with a poorer outcome. In these conditions, it may be that the NO is needed for its cytotoxic and immune functions that were recognized well before the vascular effects attributed to NO.
Although the hemoglobin solution we investigated antagonized vasodilators and had additive effects with vasoconstrictors, there was no apparent detriment to outcome in patients who received DCL-Hb after cardiac surgery (1). These early studies, by their very nature, study low-risk patient populations. The effects of the use of hemoglobin solutions in high-risk populations remain unknown. It is possible that the concerns raised by the observations above will remain theoretical and be overwhelmed by the potential benefits of hemoglobin solutions. For example, the low molecular size of hemoglobin in solution, as opposed to inside the red cell, makes for easier penetration into the interstitial space. This will provide improved tissue oxygen delivery but may be a double-edged sword by removing dilating tissue NO not otherwise bound by circulating blood, shown previously in the coronary circulation (11). It is unclear what effect hemoglobin-containing solutions may have in circumstances in which excess binding of NO may be undesirable, for example, in patients with pulmonary hypertension or after heart or lung transplantation. Moreover, there is a substantial use of inhaled NO both in critical care and after certain cardiac surgeries. The potential problem of using an intervention with significant ability to bind NO in situations in which patients are being treated with NO merits investigation.
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Acknowledgments
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This work was supported, in part, by Baxter Healthcare Corporation.
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References
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Accepted for publication September 29, 2000.
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Abstract
Introduction
