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Anesth Analg 2001;92:44-48
© 2001 International Anesthesia Research Society

CARDIOVASCULAR ANESTHESIA

Hemoglobin and Methemoglobin Concentrations After Large-Dose Infusions of Diaspirin Cross-Linked Hemoglobin

Jerome F. O’Hara, Jr, MD**, Wayne A. Colburn, PhD{dagger}, John E. Tetzlaff, MD*, Andrew C. Novick, MD{ddagger}, Kenneth W. Angermeier, MD{ddagger}, and Armin Schubert, MD, MBA*

*Department of General Anesthesiology, The Cleveland Clinic Foundation; {dagger}MDS Harris, Inc.; and {ddagger}Department of Urology, The Cleveland Clinic Foundation, Cleveland, Ohio

Address correspondence and reprint requests to Jerome F. O’Hara, Jr., MD, Department of General Anesthesiology, E31, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195.


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Diaspirin cross-linked hemoglobin (DCLHb) solution is a purified human hemoglobin product chemically stabilized to deliver oxygen to tissues. We determined the peak plasma hemoglobin concentration and assessed changes in methemoglobin concentration after the infusion of 1 g/kg DCLHb in large blood loss surgical patients. This prospective, randomized study included 26 surgical patients who were either infused with up to three 250-mL units of 10% DCLHb or transfused with up to three units of packed red blood cells during the study infusion period. Serial plasma hemoglobin, plasma methemoglobin, and whole blood methemoglobin levels were measured before and at intervals up to 48 h after the study infusion period. Plasma hemoglobin and blood methemoglobin concentrations increased during the infusion of DCLHb. The plasma hemoglobin values in the DCLHb group continued to increase during each of the infusion periods to reach a peak plasma concentration of 1450 ± 176 mg/dL. The fraction of whole blood methemoglobin increased from 0.84 ± 0.77% at baseline to 4.08 ± 1.36%. With a median DCLHb dose of 936 mg/kg (range 658–1500 mg/kg), the harmonic mean half-life was 10 h, and the increased whole blood methemoglobin reached a range not associated with complications.

Implications: The dose of diaspirin cross-linked hemoglobin (DCLHb) (936 ± 276 mg/kg) used in this study was one of the largest reported in humans to date. The DCLHb mean half-life was 10 h. The half-life observed was 2–4 times that found at smaller doses in previous studies. Whole blood methemoglobin fraction increased during DCLHb infusion but did not reach a range associated with complications.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
There is a continuous effort to develop a hemoglobin-based oxygen carrier (HBOC) that is characterized by ease of storage and transport, freedom from serious side effects, a long shelf life, compatibility with all blood types, and freedom from infectious disease risk. Widespread use of HBOCs could decrease the demand for allogenic blood donation. Currently, no HBOCs are approved for marketing by the Food and Drug Administration (FDA). Of the products evaluated in human trials, all have limited circulatory half-lives in comparison with human red blood cell transfusion (1). Diaspirin cross-linked hemoglobin (DCLHb) solution (Baxter Healthcare Corporation, Deerfield, IL) was prepared as a purified, heat-treated solution of stabilized human hemoglobin tetramers, with oxygen equilibrium characteristics similar to those of fresh red blood cells. Preclinical studies of DCLHb demonstrated oxygen transport, oxygen delivery, and volume expansion properties similar to those of whole blood, with a dose-dependent half-life (2). At a dose of 100 mg/kg, DCLHb’s half-life is 3.3 h in healthy volunteers (2) and 4.3 h in hemodialysis patients (3). No abnormalities in measured methemoglobin levels have been reported.

During a phase II clinical study, we defined the concentration of plasma hemoglobin after an 1-g/kg infusion of DCLHb and report on the methemoglobin concentration. This information is important as future HBOC solutions are developed and evaluated. Product safety, other potential beneficial effects, or the ability to decrease the need for allogenic blood transfusion are not reported.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
This prospective, randomized study was performed after obtaining IRB approval and written informed consent, along with an Investigational New Drug exemption with the US FDA. Twenty-six patients who were expected to require 3–6 U of packed red blood cells (PRBC) perioperatively were assigned to receive either DCLHb or PRBC. The first two patients received two 250-mL units of 10% DCLHb. The remaining patients were randomized to receive either three 250-mL units of 10% DCLHb (n = 12) or up to three units of PRBC (n = 12). Patients targeted for enrollment were scheduled for intraabdominal or orthopedic procedures ( Table 1). Exclusion criteria included patients with preoperative renal insufficiency (serum creatinine > 1.7 mg/dL), liver function test abnormalities (at least two tests exceeding the upper limits of normal by 50%), history of prior myocardial infarction without subsequent coronary bypass or angioplasty, or ASA physical status > III.


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  		Table 1. Surgical Procedures Performed
 
Twenty-four patients received a general anesthetic, and two patients had regional anesthesia. The actual method of anesthesia was left to the discretion of the anesthesiologist. All patients had arterial catheters and central venous pressure monitoring. Most patients who had general anesthesia also had epidural analgesia for treatment of postoperative pain. Randomization was performed, and infusion was initiated when the patient met the indication for blood administration as evaluated by the anesthesiologist assigned to the case. This generally occurred when the hematocrit was in the mid-20% range in patients without documented cardiopulmonary disease and at a hematocrit <=30% in patients with documented cardiopulmonary disease. Additional fluids (crystalloids or colloids) were infused perioperatively with the goal of maintaining blood pressure within 20% of the individual patient’s established baseline and a central venous pressure between 6 and 12 mm Hg. No additional blood products were transfused until the DCLHb units or up to three PRBCs (or autologous units plus PRBC) were administered, as per the study protocol. For a patient to remain in the study, DCLHb infusion or PRBC transfusion had to be initiated within 12 h after the start of the surgical procedure. DCLHb was administered by using a blood infusion set ( SafeLineTM ADDitIV® Primary IV Set; McGaw, Irvine, CA) with a filter ( Fenwal® 20 Micron Pediatric Transfusion Filter; Baxter Healthcare Corporation). PRBCs were administered with a standard blood filter (170- to 260-micron filter; Baxter Healthcare Corporation). The DCLHb and PRBC were warmed with conventional blood warming equipment, with the rate of infusion based on the clinical situation. DCLHb was shipped and stored at -20°C, and at least three units were kept on site for use as needed. Thawed units were kept at 2–8°C for up to 21 days and then discarded if not used. Serial plasma hemoglobin, plasma methemoglobin, and whole blood methemoglobin levels were obtained as baseline before surgery, at the end of the infusions, and at 2, 4, 6, 12, and 48 h after the study infusion period. The samples were processed and measured within 4 h of collection by using the cooximetry method of spectrophotometric determination. Data were analyzed with an unpaired Student’s t-test and Wilcoxon’s ranked sum test for median (range) values with significance set at P< 0.05. First-order elimination rate constant (Kel) was calculated by linear least-squares regression analysis using the terminal linear portion of the In (concentration) versus time curve. The terminal half-life was calculated as In(2)/Kel. Plasma concentration values were tabulated and summarized as mean ± SD by using descriptive statistics.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Twenty-six of 33 patients enrolled in the study met infusion criteria during (23 of 26) or after (3 of 26) surgery. The median DCLHb dose was 936 mg/kg (658–1500 mg/kg). The DCLHb and PRBC groups were similar with respect to age, weight, sex, preexisting cardiovascular disease, infusion indication, time needed to complete infusion, median estimated blood loss of 2100 mL (200–4500 mL) vs 2000 mL (800–3700 mL), and median total number of PRBCs—3 (1–6) vs 4 (2–11) units, respectively. Indications for infusion were continuing blood loss, a decreased hematocrit, a decrease in blood pressure, or a combination of these. In a separate publication, Schubert et al. (4) report the clinical outcome data. Mean (SD) plasma hemoglobin concentrations are presented in Figure 1.



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  		Figure 1. Mean (SD) plasma hemoglobin concentration data during and after diaspirin cross-linked hemoglobin (DCLHb) ({boxplus}) or packed red blood cells (PRBC) ({oplus}). Fourteen patients received DCLHb, whereas 12 patients received PRBC.

 
The mean plasma hemoglobin values in the DCLHb group continued to increase during each of the infusion periods, from 2.8 ± 2.2 mg/dL to 1450 ± 176 mg/dL at the end of the third infusion period. Once the infusion was discontinued, plasma hemoglobin concentrations declined with a harmonic mean half-life of 10 h. In contrast, the mean plasma hemoglobin in the PRBC group increased from 2.3 ± 2.0 mg/dL to 7.0 ± 4.3 mg/dL at the end of the first period but did not increase any more during the subsequent transfusion periods. Plasma hemoglobin values began to return toward preinfusion values after about 2 h.

In the DCLHb group, methemoglobin increased during the infusion period and persisted up to 48 h after the end of the infusion, with only a mild overall increase in the PRBC group observed. Whole blood methemoglobin as a percentage of whole blood hemoglobin in the DCLHb group was 0.84% ± 0.77% at baseline, increasing to 4.08% ± 1.36% at the end of the infusion period. However, maximal values in the DCLHb group were not achieved until 6 to 12 h after stopping the infusion in 5 of 14 patients. The largest methemoglobin whole blood fraction observed in any one patient (baseline fraction = 2.40%) was 6.10%. Methemoglobin concentrations are presented in Figure 2.



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  		Figure 2. Mean (SD) whole blood methemoglobin percentage data during and after diaspirin cross-linked hemoglobin (DCLHb) ({boxplus}) or packed red blood cells (PRBC) (<oplus>). Fourteen patients received DCLHb, whereas 12 patients received PRBC.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
At a median dose of 936 mg/kg (658–1500 mg/kg) of DCLHb, the mean peak plasma concentration observed was 1.5 g/dL, with a harmonic mean half-life of 10 hours when administered in a setting of large blood loss surgery. The infusion input function for the drug is zero order, and the pharmacokinetics are first order. An increase in whole blood methemoglobin concentration occurred in patients after infusion of either DCLHb or transfusion of PRBC but was larger in the DCLHb group. Whole blood methemoglobin remained increased for up to 48 hours after completion of DCLHb infusion. Methemoglobin was generally <6% of the total blood hemoglobin. The clinical setting of this study could not represent a pure pharmacokinetic model, considering the variable blood loss rate that occurred along with a variable volume infusion rate of crystalloids, colloids, and PRBC; therefore, peak levels of plasma hemoglobin were reported.

Ten percent DCLHb is formulated to contain <5% methemoglobin (Baxter, Hemoglobin Therapeutics). A clinical trial using 10% DCLHb found the blood methemoglobin concentration in the test solutions to be <=2.9% (2). With the lack of enzymes such as nicotinamide adenine dinucleotide-methemoglobin reductase in the stored product of DCLHb, it undergoes oxidation, forming methemoglobin as the hemoglobin converts from a ferrous to a ferric state. Factors that can increase the formation of methemoglobin with DCLHb include prolonged frozen storage and a thawed product. The rate of methemoglobin increase of DCLHb is 0.5% per month at -20°C, 1.4% per week at 4°C, and 0.4% per hour at 25°C (Baxter, Hemoglobin Therapeutics).

Mild cases of methemoglobinemia are usually treated with supportive care and the withdrawal of the toxin responsible. In more severe cases, IV 1% methylene blue at a dose of 1 to 2 mg/kg over 5 to 10 minutes should be administered (5,6). No patients in this study required treatment.

The longer half-life observed in our study most likely reflects the larger dose used. Studies in healthy subjects and chronic hemodialysis patients who used doses of 25, 50, and 100 mg/kg exhibited a prolongation in half-life (2.1–4.3 hours) as the dose increased (2,3). In a trauma study, Sloan et al. (7) infused the equivalent of 714–1428 mg/kg of DCLHb to a 70-kg person (mean [SD] number of 250-mL units was 2.5 [0.9] per patient) but did not report on plasma hemoglobin concentrations, methemoglobin concentrations, or the plasma half-life of the product (7). That study reported an increased mortality for DCLHb-treated patients. Recently, Lamy et al. (8) reported that infusion of up to 750 mL of 10% DCLHb allowed 19% of cardiac surgery patients (versus no control patients) to avoid exposure to allogenic blood after surgery. In that study, patients who received the maximum dose of DCLHb (750 mL), the peak mean plasma hemoglobin value was 1.3 gm/dL, which became less than one-half the peak value at 24 hours after the start of the first infusion. Methemoglobin concentrations were not reported.

The pharmacokinetic profile of DCLHb in the current study could have been affected by continuing blood loss and the need to administer colloid, crystalloid, or additional blood products. This affect could have increased the volume of distribution for DCLHb by simple dilution but should not have affected the directly measured half-life.

The tendency for whole blood methemoglobin to increase even after DCLHb infusion is discontinued indicates that there is a delay in reaching maximal methemoglobin concentrations in whole blood. Maximum observed whole blood methemoglobin values occurred well after the final infusion was completed and increased more dramatically than whole blood percentages as plasma hemoglobin concentrations decreased after discontinuation of DCLHb administration. It is likely that methemoglobin percentages calculated on the basis of total whole blood hemoglobin concentrations became artifacts of the computational and analytical methods used to determine the percentages as concentrations approached zero. At 48 hours, plasma hemoglobin concentrations in the DCLHb group were near those of the PRBC group, but methemoglobin concentrations remained increased. The exact length of time that methemoglobinemia was increased in the DCLHb group cannot be determined from this study.

The increased half-life of DCLHb infusion observed in this study may be of limited clinical benefit when compared to the longer intravascular life span of PRBC transfusion. Current HBOC solutions might be considered for infusion during acute exsanguination and for a bridge to transfusion while an appropriate cross-match is performed, or it might be infused in certain patient populations to actually avoid allogenic blood transfusions (8). For HBOCs to assume more of a clinical role, they likely will need to be manufactured to have a longer half-life.

The clinical significance of this study is to report the half-life of DCLHb (currently a discontinued product) at this study dose and to report that methemoglobinemia occurred. We concluded that peak plasma hemoglobin concentrations of DCLHb in large blood loss surgery patients were similar to those reported in postoperative cardiac patients receiving a comparable dose. The mean increase in plasma hemoglobin after 1 gm/kg of DCLHb infusion in this study was 1.5 gm/dL, and the harmonic mean half-life was 10 hours. The increase in methemoglobin in the DCLHb patient group was less than the level associated with cyanosis and appeared to be clinically insignificant under the conditions studied. Future generation HBOC solutions will ideally provide a longer half-life and need to be evaluated for an increased methemoglobin concentration. Product safety, other potential beneficial effects, or the ability to decrease the need for allogenic blood transfusion are not reported.


   Acknowledgments
 
This study was supported by a grant from Baxter Healthcare Corporation, Hemoglobin Therapeutics Division.


   Footnotes
 
Presented in part at the Annual Meeting of the American Society of Anesthesiologists, San Diego, CA, October, 1997.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Dietz NM, Joyner MJ, Warner MA. Blood substitutes: fluids, drugs or miracle solutions? Anesth Analg 1996; 82: 390–405.[Abstract]
  2. Przybelski RJ, Daily EK, Kisicki JC, et al. Phase I study of the safety and pharmacologic effects of diaspirin cross-linked hemoglobin solution. Crit Care Med 1996; 24: 1993–2000.[ISI][Medline]
  3. Swan SK, Halstenson CE, Collins AJ, et al. Pharmacologic profile of diaspirin cross-linked hemoglobin in hemodialysis patients. Am J Kidney Dis 1995; 26: 918–23.[ISI][Medline]
  4. Schubert A, O’Hara JF Jr, Przybelski RJ, et al. Effect of diaspirin crosslinked hemoglobin (DCLHb HemAssist TM) during high blood loss surgery on selected indices of organ function [abstract]. Anesthesiology 1997; 87: A220.
  5. Coleman MD, Coleman NA. Drug-induced methemoglobinemia. Drug Saf 1996; 14: 394–405.[ISI][Medline]
  6. Laney RF, Hoffman RS. Methemoglobinemia secondary to automobile exhaust fumes. Am J Emerg Med 1992; 10: 426–8.[Medline]
  7. Sloan EP, Koenigsberg M, Gens D, et al. Diaspirin cross-linked hemoglobin (DCLHb) in the treatment of severe traumatic hemorrhagic shock. JAMA 1999; 282: 1857–64.[Abstract/Free Full Text]
  8. Lamy ML, Daily EK, Brichant JF, et al. Randomized trial of diaspirin cross-linked hemoglobin solution as an alternative to blood transfusion after cardiac surgery. Anesthesiology 2000; 92: 646–56.[ISI][Medline]
Accepted for publication September 25, 2000.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press