Peter C. Canizaro

Nephrotoxicity of Hemoglobin Solutions

Nephrotoxicity is a problem of hemoglobin solutions (HbS) that still awaits full elucidation and correction. Therefore, a study was conducted using five HbS with different characteristics to replace 1/3 of blood volume in five groups of rabbits. All HbS contained bovine Hb, 6.5 g/dl, dissolved into a balanced electrolyte solution. HbS-I was Hb incompletely purified of stromal phospholipids and environmental bacterial endotoxins, and uncrosslinked; HbS-II was pure Hb non crosslinked; HbS-III was completely purified and crosslinked; HbS-IV was like HbS-III, but with pH 8.4; and HbS-V was like HbS-III, with the addition of mannitol. The effects of blood replacement with these solutions were studied on: (a) PAH clearance (expression of renal plasma flow); (b) endogenous creatinine clearance (expression of glomerular filtration); (c) fractional excretion of sodium and (d) urine/plasma osmolarity (expressions of tubular function). Histological changes were assessed after 24 hours. Significant alterations were observed in decrescent order following the administration of HbS-I, -II and -III, while HbS-IV and -V were well tolerated. These results suggest that the nephrotoxicity of Hb solutions can be prevented by the following steps: (1) complete purification of Hb; (2) complete crosslinking; and (3) protection of the kidney by alkalinization of the urine and/or the addition of mannitol.

Cleavage of the Arg1-Pro2 bond of bradykinin by a human lung peptidase: isolation, characterization, and inhibition by several beta-lactam antibiotics.

Abstract An aminopeptidase P (EC 3.4.11.9) that cleaves the Arg1-Pro2 bond of bradykinin has been isolated for the first time from human lung and purified 473-fold. The enzyme also catalyzes the cleavage of arginine from des-[Arg9]-bradykinin and the hydrolysis of several X-proline dipeptides including L-arginyl-L-proline, L-leucyl-L-proline, and L-alanyl-L-proline. Purified enzyme was routinely assayed (after initial identification with des-[Arg9]-bradykinin) with L-leucyl-L-proline. The molecular weight, in nondenaturing buffers, is 188,000 ± 8500 Da. The pH optimum was 8.0 with arginyl-proline, and was 6.8 with leucyl-proline. Chelating agents do not inactivate the enzyme, but rather only remove loosely bound cations that stimulate the enzyme. Manganese is the principal cation that stimulates the enzyme. The enzyme is inhibited by several β-lactam antibiotics, cephalexin and oxacillin being the most effective of those tested. The antibiotic inhibition is time and temperature dependent, and it is not fully reversible by exhaustive dialysis of the antibiotic-treated enzyme.

Toxic factors in the red blood cell membrane.

The toxic effects of hemolysed RBCs have been studied for more than 100 years, but the specific factors involved have not been identified. This study focused on phosphatidylethanolamine (PE) and phosphatidylserine (PS), two aminophospholipids that normally reside on the cytoplasmic side of the red cell membrane. An in vitro experiment with murine peritoneal exudate macrophages showed that PE and PS: a) stimulated the production of H2O2, complement factor C3a, prostacyclin, and thromboxane at a dose of 5 micrograms/ml; b) produced cell injury, evidenced by release of lipid peroxides, LDH, and by morphologic changes on phase-contrast and electron microscopy at a dose of 50 micrograms/ml; and c) caused cell death in 50-66% of cells at a dose of 100 micrograms/ml. An in vivo experiment showed that PE and PS injected intravenously into various groups of rabbits: a) caused only transient hypotension at a dose of 0.05 mg/kg body weight; b) caused significant hypotension, cardiac arrhythmias, bronchospasm, activation of intravascular coagulation, complement, platelets, and leukocytes with release of histamine, serotonin, and thromboxane at a dose of 0.10 mg/kg; and c) caused cardiac arrest and death at a dose of 0.30 mg/kg. In contrast, the phospholipids of the outer cell membrane (phosphatidylcholine and phosphatidylinositol) caused minimal toxicity in vitro and none in vivo.

Evaluation of anion-exchange liquid chromatography for purification of hemoglobin from peptides and other proteins.

Abstract Solutions of hemoglobin (Hb) are under development for use as “red blood cell substitutes”. In order to reduce the presence of immunogenic and vasoactive factors in these solutions, the elimination of non-Hb proteins and peptides is necessary. The ability of anion-exchange liquid chromatography (LC) to purify Hb, using the Waters HPLC 600E system and five different columns (HP-PEI, MA7P, DEAE-5PW, MA7Q and Mono-Q HR) was evaluated. Bovine Hb, prepared by hyposmotic dialysis-ultrafiltration of isolated red blood cells, was subjected to chromatography by each column. The major fraction (HbAo) was analyzed by re-separation through the same column and by isoelectric focusing (IEF) with silver staining. The second chromatogram revealed HbAo to contain 2–4 non-Hb contaminants, some of which adherent to and some separate from hemoglobin. IEF revealed the full spectrum of Hb contamination, with 3–5 protein bands plus 10–15 peptide bands in association with HbAo. The results suggest that anion-exchange cannot completely purify hemoglobin and IEF with silver staining is more sensitive than LC.