Hemolink-induced effects on intestinal motor function and attenuation of these effects by selected agents.

Hemolink, an oxidized, ring-opened raffinose-crosslinked hemoglobin-based oxygen carrier produced by Hemosol Inc., stimulates esophageal peristalsis, possibly by interference with neural NO-mediated effects. The effects of Hemolink on jejunal tone and contractions, arterial pressure and heart rate were measured in anesthetized rats, and the effect of selected agents in attenuating or reversing these effects was studied. Infusion of L-NAME was used to validate the study model; it caused an immediate increase in tone and initiated phasic contractions indicating that the model was responsive to NO-mediated effects. Hemolink administration caused effects on intestinal motor function similar to those caused by L-NAME, including increases in basal tone and contraction amplitude. Rat whole blood caused none of these changes. The Hemolink-induced effects were less immediate in some animals compared to those observed after L-NAME. As well there was greater inter-animal variability on the effects. Hemolink administration also caused a mild increase in arterial blood pressure and a reciprocal decrease in heart rate in some animals. Co-administration of morphine, a common analgesic that has been reported to influence the motility of the GI tract; L-arginine, a substrate for NO synthesis; and glycopyrrolate, an anti-cholinergic agent, did not significantly modulate the Hemolink effects, whereas nitroglycerin, an NO donor; and nifedipine, a slow calcium-channel blocker, attenuated or reversed these effects.

Pharmacokinetic studies in the rat on a o-raffinose polymerized human hemoglobin.

We have studied the plasma half-life (T 1/2), oxygen-binding affinity (P50), organ distribution, and excretion of the individual molecular weight (MW) components of human hemoglobin polymerized with periodate-oxidized, ring-opened raffinose (oR poly-Hb), following transfusion in the rat. The model was an isovolemic 50% exchange transfusion in the conscious, chronically catheterized rat. Total plasma Hb levels yielded a (T 1/2) of 10 to 11 hr for oR poly-Hb. The T 1/2 values of individual MW components of the poly-Hb as determined by size-exclusion HPLC were approximately: 4 hr for the monomeric fraction (Hb)1, 9 hr for the dimer (Hb)2, and 15 hr for the fraction representing trimers to nanomers (Hb)3-9. The P50 values of plasma samples containing oR poly-Hb (collected from 0-24 hr after exchange) remained unchanged at 28 +/- 3 mmHg. oR stabilized and polymerized Hb were not excreted via the kidneys. Hepatic and renal distribution as well as plasma and renal clearance were determined by liquid scintillation counting using individual tritium [3H] labelled MW components purified from [3H]-oR poly-Hb: (Hb)1/2, (Hb)1, (Hb)2, (Hb)3&4, and (Hb) greater than 9. In kidney, uptake (determined by the relative concentration of radioactivity) decreased with increasing MW of the labelled component. Conversely, in liver, uptake increased with increasing MW. Plasma and renal clearance results were consistent with those obtained by HPLC analysis. Hematocrit levels returned from a 20% post-transfusion level to normal pre-transfusion levels (44%) within 10 days after the exchange.

Quality control of hemoglobin-based blood substitutes.

Preparation and use of hemoglobin-based blood substitutes, from stroma-free hemoglobin (SFH or Hb) and its DPG analogue-modified derivatives (PLP-Hb, ATP-Hb etc.) without thorough characterization and quality control in animal or human testing have produced, and may continue to produce, artifacts in the finished product. Thus the development of such a natural substitute for the volume expansion and oxygen delivery functions of the blood will be impeded. A case is made for the use of affinity purified hemoglobin and modified hemoglobin as standard starting materials for the preparation of Hb-based blood substitute(s) in general, and in particular poly PLP-Hb. Development of a commercial scale blood-substitute is only possible after the safety and toxicity issues of substitutes have been resolved by applying rigorous quality control.

Hemoglobin-based blood substitutes: characterization of five pyridoxal 5'-phosphate derivatives of hemoglobin.

Six fractions (I-VI) of pyridoxal 5'-phosphate (PLP) hemoglobin (Hb), prepared by the method of De Venuto and Zegna [J. Surg. Res., 34 (1983) 205] have been isolated and purified by anion-exchange high-performance liquid chromatography (HPLC). Total phosphate analyses indicate that I is HbA, II and III are double-labelled, IV and V are tetra-labelled and VI contains 6 mol of phosphorus per mol of hemoglobin. The purified components have been resolved into their alpha and beta chains by preparative reversed-phase HPLC using a macroporous C4 support. Phosphate analyses indicate that the beta chains of II, III and IV each contain one phosphate per chain while the beta chains of V and VI each contain two phosphates. The alpha chains of IV and VI were found to be monophosphate-labelled. Reversed phase HPLC analysis of the tryptic peptides of the beta chains indicates that the label is bound exclusively to the 1-valine residue in II, III and IV while both the 1-valine and the 82-lysine are labelled in V and VI. Similarly, modification of the 1-valine residue of the alpha chains of IV and VI was detected. Components II and III have the same molecular formula. Evidence is presented which shows that they are interconvertible and that they correspond to the PLP2-Hb species and component V is Benesch's PLP4-Hb [J. Biol. Chem., 257 (1983) 1320 and references cited therein]. Component IV is III with one additional PLP per alpha chain and similarly VI is V with monolabelled alpha chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced coronary vasoconstrictor activity of hemoglobin solutions purified by ATP-agarose affinity chromatography.

Stroma-free hemoglobin (Hb) solutions are being developed as blood substitutes. We previously described coronary vasoconstrictor activity of Hb solutions prepared by conventional methods. In the present study we assessed the constrictor activity of unmodified and covalently modified Hb solutions purified by ATP-agarose affinity chromatography. The starting material was a red cell lysate, partially purified by ultrafiltration. Coronary constrictor activity was measured as increased perfusion pressure in isolated rabbit hearts perfused at constant coronary flow rate with buffer containing various concentrations of added Hb. The starting material increased perfusion pressure by 35 +/- 7 mmHg at 50 mg/dl. Purified Hb, retained by the affinity column, increased perfusion pressure by only 18 +/- 2 mmHg at 50 mg/dl. Hemoglobin covalently linked to ATP or pyridoxal phosphate, then purified by affinity chromatography, also had less constrictor activity than the starting material. Thus, a substance, removed by affinity chromatography but not by conventional purification, contributes to the vasoconstrictor activity of Hb solutions.

Isolation and partial characterization of pyridoxal 5'-phosphate hemoglobins by high-performance liquid chromatography as a quality-control method for hemoglobin-based blood substitutes.

Pyridoxal 5'-phosphate hemoglobin (PLP-Hb), prepared from hemoglobin and a four-fold excess of pyridoxal 5'-phosphate by the method of De Venuto and Zegna [J. Surg. Res., 34 (1983) 205], has been chromatographically resolved into six components via a quaternary ammonium monobead support. On an analytical scale, the separations have been found to be rapid (ca. 50 min) and highly reproducible. The results also indicate that the preparation of PLP-Hb yields a reproducible product ratio. The potential of the analytical method for the routine quality control of blood substitutes derived from PLP-Hb is discussed. All five of the PLP derivatives (components II-VI), isolated and purified via a combination of conventional and preparative monobead anion-exchange chromatography, gave single peaks when analyzed by high-performance liquid chromatography. Total phosphate analyses indicated that components II and III each contain two PLPs per Hb, IV and V four and VI six.

Bovine serum albumin: characterization of a fatty acid binding site on the N-terminal peptic fragment using a new spin-label.

A new spin-label, 4-(L-glutamo)-4'-[(1-oxy-2,2,5,5-tetramethyl-3L-pyrrolidinyl )amino]-3, 3'-dinitrodiphenyl sulfone, is shown to bind to one high-affinity binding site on bovine serum albumin (K = 5 X 10(4) M-1, n = 1). Analysis of the binding of the spin-label to the amino-terminal half (peptic fragment PB) and the carboxy-terminal half (peptic fragment PA) of BSA, and their complex (PA-PB), indicates that the spin-label binds to a long-chain fatty acid binding site located on PB. The usefulness of the novel specificity of the spin-label in characterizing this binding site is discussed.

alpha-fetoprotein binding specificity for arachidonate, bilirubin, docosahexaenoate, and palmitate. A spin label study.

A dianionic spin label, 1-L-glutamate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-i,4-dinitrobenzene, has been used to probe the relative binding specificity of a single anionic ligand site on bovine alpha-fetoprotein (AFP) to arachidonate, bilirubin, docosahexaenoate, and plamitate. The binding isotherm of the spin label with AFP, as shown by a Scatchard plot, indicates the presence of a single high affinity binding site. The site-site relationship of the four endogenous ligands, arachidonate, bilirubin, docosahexaenoate, and palmitate, was determined by studying their effectiveness in competing for this anionic ligand binding site on AFP. Scatchard plots of the spin label in the presence of 1 to 3 molar equivalents of arachidonate, bilirubin, and docosahexaenoate and up to 6 molar equivalents of palmitate have been determined. The effectiveness of the four endogenous ligands in displacing the spin label from its primary binding site is bilirubin greater than or equal arachidonate approximately equal to docosahexaenoate greater than palmitate. These results indicate that polyunsaturated essential fatty acids and bilirubin share a high affinity binding site on AFP. We propose that the function of this anionic ligand binding site on AFP is for the transport of bilirubin and polyunsaturated fatty acids in fetal serum, as well as for the cross-placental transfer of this metabolite and of essential fatty acids.

Development of a spin assay for reserve bilirubin loading capacity of human serum.

The capacity of human serum albumin to bind bilirubin is of paramount importance in the prevention of neurological damage due to hyperbilirubinemia in the neonate. Currently, there is no rapid, reliable method to determine the reserve bilirubin loading capacity of serum albumin. A method using electron spin resonance spectroscopy with a dianionic spin-label molecular probe [1-N-(2,2,6,6-tetramethyl-1-oxyl-4-piperidinyl)-5-N-(1-aspartate)-2,4-dinitrobenzene] is presented which yields the reserve binding capacity of the bilirubin high-affinity binding site on human serum albumin and reserve bilirubin loading capacity of serum in mg/dl. Clinical validation of this spin assay may improve the neonatal care of jaundiced newborns.