Abstract
Hemoglobin based oxygen carriers (HBOC) are resuspended in “excipients” consisting of Ringer's D,L-lactate containing antioxidants to prevent methemoglobin formation during storage.
Investigators have reported cardiac arrhythmias following infusion of Ringer's D,L-lactate solution. Studies have shown that D-lactate stimulates human granulocytes to generate oxygen free radicals and L-lactate inhibits glycolysis.
Patients receiving HBOC in Ringer's D,L-lactate excipient are also resuscitated or hemodiluted with Ringer's lactate solution. Oxygen-free radicals generated by Ringer's D,L-lactate and HBOC may oxidize nitric oxide in endothelial cells, causing the vasoconstrictor effects reported following HBOC infusion, and activate NF-κb and the apoptotic cascade. The combination of Ringer's D,L-lactate and HBOC in Ringer's D,L-lactate excipient may be responsible for the severe adverse events observed in clinical studies of HBOC.
Veech has recommended replacing the 27 mM of lactate in Ringer's with 27 mM D-betahydroxybutyrate (BHB). BHB reduces the generation of oxygen free radicals by mitochondria and human granulocytes.
INTRODUCTION
Hemoglobin based oxygen carriers (HBOC) have been resuspended in media, referred to as excipients, for storage at 22° C or 4° C for as long as 2 years prior to infusion. The excipient for the HBOC contains Ringer's D,L-lactate supplemented with antioxidants to prevent the formation of methemoglobin during storage.
BACKGROUND
Toxicity of Ringer's D,L Lactate—The Principle Resuscitation Fluid for the Past 50 Years
At the Institute of Medicine meeting held in September 1998 in Washington, DC, on resuscitative fluids, Dr. Richard L. Veech reported on the toxicity of Ringer's D,L lactate [Citation[1]]. Dr. Veech reported on the cardiac arrhythmias in experimental animals and in patients infused with Ringer's D,L lactate solution [Citation[2]]. He also reported that D lactate generated oxygen free radicals and that the metabolism of L-lactate inhibited glycolysis. Dr. Veech recommended the use of Ringer's ketone solution in which 27 mM D-betahydroxybutyrate is substituted for the the 27 mM of D,L lactate. Subsequent studies by Dr. Hasan Alam and his associates [Citation[3] Citation[4]] reported that resuscitation induced pulmonary apoptosis and intracellular adhesion molecule-1 expression in rats produced by Ringer's D,L lactate were attenuated by the use of Ringer's ketone solution and Ringer's pyruvate solution, Ringer's ketone solution is stable at room temperature for at least 2 years, whereas Ringer's Na pyruvate and Ringer's ethyl pyruvate solutions are not stable at room temperature.
Studies during the Vietnam War showed that the resuscitation of young wounded servicemen with large volumes of crystalloid solution consisting of Ringer's D,L lactate was associated with acute respiratory distress syndrome referred to as the “Danang Lung.” Recent studies in which rats subjected to hemorrhagic shock were resuscitated with Ringer's D,L lactate demonstrated the toxic effect of Ringer's D,L lactate in the production of pulmonary, liver and intestinal apoptosis [Citation[3-8]]. Rhee and associates [Citation[9-11]] have reported that Ringer's D,L lactate stimulates human granulocytes to produce oxygen free radicals and that Ringer's L lactate has less of an effect in the stimulation of human granulocytes to produce oxygen free radicals.
The documentation of the toxicity of Ringer's D,L lactate and the demonstration that the D isomer of lactate was primarily responsible for its toxicity resulted in the modification of the Ringer's D,L lactate solution with no clinical testing. The solution now contains only the L isomer of lactate, i.e., Ringer's L-lactate. However, Cross and associates [Citation[12]] have confirmed that L-lactate inhibits glycolysis and impairs resuscitation of the heart.
Toxicity of Hemoglobin Based Oxygen Carriers (HBOC)
The toxicity of hemoglobin based oxygen carriers has been related to infectious disease and the immunogenicity of bovine and human hemoglobin, endotoxin contamination, residual RBC stroma, molecular weight of the hemoglobin species—percentages of dimer, tetramer, and polymerized hemoglobin, the cross-linking agent—glutaraldehyde and O-raffinose, oxygen affinity (p50) of hemoglobin based oxygen carrier, viscosity, colloid-osmotic pressure, and level of methemoglobin. HBOC have been shown to generate oxygen free radicals depending on their affinity for oxygen—the higher the p50, the greater the ability of the HBOC to generate oxygen free radicals.
It has been suggested that hemoglobin solutions may exacerbate ischemia-reperfusion injury [Citation[13] Citation[14]]. The term reperfusion injury refers to the oxidant injury that is caused by the generation of reactive oxygen species after blood flow is restored to an ischemic organ (i.e. heart, lung, liver, intestine or kidney) [Citation[15] Citation[16]]. Hemoglobin could theoretically contribute to reperfusion injury in at least two ways: (1) by providing additional oxygen, a necessary substrate for reactive oxygen species formation; and (2) by providing a source of free iron, which acts as an essential catalyst in the formation of the hydroxyl radical [Citation[17]]. The oxygen free radicals produced by HBOC may oxidize endothelial nitric oxide, which may explain in part the observed vasoconstrictor effects of HBOC. In addition, the generation of free radicals activate NF-κb and the apoptotic cascade [Citation[18-20]].
Combined Toxicity of Ringer's D,L Lactate and HBOC
The toxicity of the Ringer's D,L lactate, which has been used as the principle resuscitation fluid used in clinical medicine and as the excipient for the HBOC for more than 30 years, may augment the toxicity of the HBOC. Both Ringer's D,L lactate and the HBOC produce oxygen free radicals, and the presence of both in the patient may be responsible for the severe adverse events that have been observed. Some patients receiving HBOC in the Ringer's D,L lactate excipient are also being resuscitated with Ringer's D,L lactate solution, and patients undergoing cardiopulmonary bypass surgery may also be receiving Ringer's D,L lactate to produce hemodilution during the procedure. It has been reported that diabetic patients have a higher incidence of severe adverse events than non-diabetic patients following cardiopulmonary bypass surgery when they received HBOC in the Ringer's D,L lactate excipient, and in these patients, too, Ringer's D,L lactate may be infused as the resuscitative solution and as the hemodilution solution.
CONCLUSION
The resuscitative fluids and the excipient of the HBOC should be able to tranquilize the oxygen free radicals that may be released by the HBOC. Betahydroxybutyrate, which replaces the D,L lactate in the Ringer's ketone solution, has been shown to attenuate the pulmonary apoptosis in rat lungs following hemorrhagic shock and resuscitation [Citation[3] Citation[4] Citation[8]]. In addition, betahydroxybutyrate reduces the generation of oxygen free radicals by mitochondria and by human granulocytes [Citation[21] Citation[22]]. Rats subjected to hemorrhagic shock and resuscitated with Ringer's ketone solution had significantly better survival and myocardial function than rats resuscitated with Ringer's D,L lactate (Apstein et al., to be published). Hypovolemic-anemic rats subjected to renal ischemia and resuscitated with Ringer's ketone solution had significantly better renal function 48 hours after resuscitation than rats treated with Ringer's D,L lactate solution (Lieberthal et al., to be published). These studies demonstrated that Ringer's ketone is superior to Ringer's D,L lactate as a resuscitative fluid.
The data indicate that the combination of the resuscitative fluid and HBOC containing Ringer's D,L lactate may have been responsible for the reported severe adverse events observed in clinical studies of HBOC. Ringer's D,L lactate has been shown to be toxic and should not be used as the excipient for HBOC. Further investigation is necessary to determine the safety of Ringer's L lactate both as a resuscitative fluid and as an excipient for the HBOC. The therapeutic effectiveness of Ringer's ketone solution in the resuscitation of animals has been demonstrated, making it an ideal resuscitative fluid and excipient for the HBOC.
This work was supported by the U.S. Navy (Office of Naval Research Contract N00014-02-1-0923). The opinions or assertions contained herein are those of the authors and are not to be construed as official or reflecting the views of the Navy Department or Naval Service at large.
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