Synthesis and characterization of a novel DTPA polymerized hemoglobin based oxygen carrier.
OBJECTIVE
The purpose of this study was to prepare a novel polymerized hemoglobin (Hb) based oxygen carrier (HBOC) designed to minimize Hb induced hypertension, while employing a simple and inexpensive method of preparation. Cyclic-diethylenetriaminepentaacetic acid (DTPA) was used to polymerize stroma free Hb (SF-Hb).


METHODS
SF-Hb was isolated from red blood cells and reacted with DTPA at a constant concentration, pH, and duration. Low molar mass fractions (<100 kDa) were removed using ultrafiltration. Reactions and subsequent ultrafiltration steps were determined to be reproducible by analyzing molar mass, colloid osmotic pressure and oxygen affinity. Finally, a model of 50% exchange transfusion (ET) in rats was used to evaluate the blood pressure response to DTPA polymerized SF-Hb (Poly-DTPA-Hb).


RESULTS
Poly-DTPA-Hb demonstrated a number averaged molar mass of 128.7 kDa and a weighted average of 223.0 kDa. Oxygen binding equilibrium indicated high oxygen affinity (P50 = 5.1+/-0.01 mm Hg) and little cooperativity (n = 1.4). Poly-DTPA-Hb and a control DTPA polymerized human serum albumin (Poly-DTPA-HSA) unexpectedly caused acute hypotension during the period of ET in rats (mean arterial pressure approximately 45% less than baseline). Hypotension occurring over the period of ET was determined to be mediated by calcium binding to protein associated DTPA. This effect was attenuated by the addition of calcium chloride (CaCl2) to the Poly-DTPA protein preparations.


CONCLUSIONS
Cyclic DTPA anhydride can be used to create cross-linked and polymerized hemoglobin, using a simple and inexpensive process. However, the addition of CaCl2 to the preparation appears to be required to prevent calcium chelation and subsequent hypotension during infusion.
Haney
Chad R
CR
Department of Bioengineering, The University of Illinois at Chicago, Chicago, IL 60612, USA. chaney@rover.bsd.uchicago.edu
Buehler
Paul W
PW
Gulati
Anil
A
D000818:Animals; D001794:Blood Pressure / Q000187:drug effects; D001802:Blood Substitutes / Q000138:chemical synthesis*; D002850:Chromatography, Gel; D005078:Exchange Transfusion, Whole Blood; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D007834:Lasers; D008297:Male; D008970:Molecular Weight; D009391:Nephelometry and Turbidimetry; D009997:Osmotic Pressure; D010100:Oxygen / Q000097:blood; D004369:Pentetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001802:Blood Substitutes; D006454:Hemoglobins; C016484:hemoglobin, stroma free; C039777:cyclic DTPA anhydride; D004369:Pentetic Acid; D010100:Oxygen