Bovine Hemoglobin Solution Research Articles

Bovine hemoglobin increases skeletal muscle oxygenation during 95% artificial arterial stenosis

Background . This study investigates the effect of a stroma-free ultmpurified bovine hemoglobin solution (HBOC) on skeletal muscle tissue oxygenation in comparison with hetastarch during nearly complete arterial stenosis. Methods . Fourteen foxhounds were intravenously anesthetized and mechanically ventilated with 30% oxygen in air. Catheters were inserted into the right femoral artery and vein for measurements of hemodynamic parameters and blood gas sampling. Arterial blood flow of the left popliteal artery was measured by means of an electromagnetic flow probe. Skeletal muscle tissue oxygen tension (tpO 2) was measured in the left gastrocnemius muscle by using a stepwise driven polarographic needle probe creating histograms from 200 single tpO 2 measurements. After isovolemic hemodilution with Ringer's lactate solution to a hematocrit of 25%, a 95% artificial stenosis of the popliteal artery was established. The animals then randomly received two applications of either 50 ml HBOC (molecular weight, 32,000 to 500,000; hemoglobin, 13 ± 1 gm/dl −1) or 200 ml 6% hetastarch 200,000/0.5. Variables were measured at baseline, after hemodilution, 30 minutes after stenosis, and 15 minutes after two applications of the respective compound. Results . Demographic data, muscle temperature, and arterial blood gases did not differ between groups. With the exception of higher mean arterial and mean pulmonary artery pressures in HBOC-treated animals, hemodynamics did not differ between groups. In both groups oxygen delivery and oxygen consumption of the muscle decreased in parallel to the decreasing blood flow during arterial stenosis. In contrast, oxygen extraction ratio increased after infusion of HBOC and was higher after the second application when compared with hetastarch-treated animals (p < 0.05). During stenosis tpO 2 was decreased in both groups when compared with baseline (p < 0.001). Mean tpO2 remained at decreased levels after administration of hetastarch but increased to nearly baseline values after HBOC treatment (p < 0.001). Conclusions . The data suggest that increased oxygen extraction in the HBOC group is associated with improved skeletal muscle tissue oxygenation during severe arterial stenosis.

Submicron polymer membrane hemoglobin nanocapsules as potential blood substitutes: preparation and characterization.

Biodegradable polymer membrane nanocapsules containing hemoglobin have been prepared with phase separation and polymer precipitation methods. We have used different polymers including polylactic acid and polyisobutyl-cyanoacrylate. The mean diameter of the hemoglobin nanocapsules can be as low as 0.07 mu and up to 0.39 mu dependent on the method of preparation. The concentration of the hemoglobin in the suspension can be up to 11 gm/dl, as compared to 15 gm/dl in whole blood. The viscosity of the preparation is slightly lower than that of human blood. In vitro tests show that P50 of these nanocapsules containing bovine hemoglobin is identical to free bovine hemoglobin solution. The surface of the nanocapsules can be modified for example with polyethylene glycol(PEG) or with PEG 2000 PE. Preliminary in vivo tests show that surface modified nanocapsules containing hemoglobin can survive longer in the circulation.

Administration of a hemoglobin solution decreases hypoxia and increases radiation response in a rat mammary carcinoma

AbstractAdministration of an ultrapurified bovine hemoglobin solution (8 ml/kg, iv) to female Fisher 344 rats bearing the 13672 mammary carcinoma implanted subcutaneously in the hind leg reduced the hypoxic fraction of the tumor from 49 to 36% with normal air breathing and to 28% with carbogen (95% oxygen/5% carbon dioxide) breathing. When administration of the hemoglobin solution was followed by single dose (10, 20, or 30 Gray) local radiation therapy, the dose modifying factor was 1.5 with air breathing and 2.7 with carbogen breathing. For fractionated radiation therapy, 2, 3, or 4 Gray was delivered locally daily for 5 days. When the hemoglobin solution was administered prior to each radiation fraction and air breathing maintained, there was a 1.8‐fold increase in tumor growth delay. When daily hemoglobin solution administration was accompanied by carbogen breathing, there was a 2.6‐fold increase in tumor growth delay. Less frquent administration of the hemoglobin solution (alternate days, twice weekly or once weekly) resulted in 1.5‐ to 1.7‐fold increases in tumor growth delay. The greatest increases in tumor oxygenation occurred shortly after administration of the hemoglobin solution; in fact at 12 h post administration of the hemoglobin solution, the tumors appeared to be more hypoxic than prior to administration of the hemoglobin solution. By 24 h post administration of the hemoglobin solution, the oxygen tension profile of the tumors were returning to pretreatment levels. Further investigation of this ultrapurified hemoglobin solution in cancer is warranted. © 1995 Wiley‐Liss, Inc.

Intermolecular electrostatic interactions and their effect on flux and protein deposition during protein filtration.

Although membrane filtration is used extensively to process protein solutions containing a variety of electrolytes, there is currently little fundamental understanding of the effect of the solution environment (and in particular, the solution pH) on the filtrate flux in these systems. We have obtained data for the flux and sieving coefficients during the batch (stirred cell) filtration of solutions of bovine serum albumin, immunoglobulins, hemoglobin, ribonuclease A, and lysozyme through 0.16-micron microfiltration membranes at different pH values. The flux declined significantly for all five proteins due to the formation of a protein deposit on the upper surface of the membrane. The quasi-steady ultrafiltrate fluxes at the individual protein isoelectric pH's were essentially identical, despite the large differences in molecular weight and physicochemical characteristics of these proteins. The flux increased at pH's away from the isoelectric point, with the data well-correlated with the protein surface charge density. These results were explained in terms of a simple physical model in which the protein deposit continues to grow, and thus the flux continues to decline, until the drag force on the proteins associated with the filtrate flow is no longer able to overcome the intermolecular repulsive interactions between the proteins in the bulk solution and those in the protein deposit on the surface of the membrane.

Oxygenation of the Rat 9l Gliosarcoma and the Rat 13672 Mammary Carcinoma with Various Doses of a Hemoglobin Solution

Tumor oxygen tensions were measured using a computer controlled pO2 microelectrode in two preclinical solid tumor models, the rat 9L gliosarcoma and the rat 13672 mammary carcinoma. Tumor oxygenation profiles were determined under four conditions: 1) normal air breathing, 2) carbogen (95% O2/5% CO2) breathing, 3) after intravenous administration of a solution of ultrapurified polymerized bovine hemoglobin with normal air breathing and 4) after intravenous administration of a solution of ultrapurified polymerized bovine hemoglobin with carbogen breathing. Both tumors had severely hypoxic regions under normal air breathing conditions. Although carbogen breathing increased the oxygenation of the better oxygenated portions of the tumor, it did not impact on the severely hypoxic tumor regions. Administration of the hemoglobin solution was effective in increasing the oxygenation throughout both tumors under normal air breathing conditions. The addition of carbogen breathing to administration of the hemoglobin solution eliminated severe hypoxia in the 9L gliosarcoma and markedly reduced the severely hypoxic regions of the 13672 mammary carcinoma.

Reaction of human endothelial cells to bovine hemoglobin solutions and tumor necrosis factor.

Human umbilical vein endothelial cells (HUVEC) were incubated for 24 hours with 0.1 mM or 0.3 mM of: [A] unmodified (U) Hb-FeIIO2; [B] UHb-FeIII; [C] UHb-FeIV-OH; [D] polymerized low molecular weight Hb (< 400 kDa); [E] polymerized high molecular weight Hb (< 1,020 kDa); [F] polymerized low molecular weight Hb + Endotoxin (2.5 EU/mL); [G] rTNF alpha 100 pg/mL; [H] rTNF alpha 400 pg/mL; [I] rTNF alpha 800 pg/mL. The medium of the incubation was tested for LDH (index of cell injury), and for cytokines GM-CSF and IL-1 alpha released by the cells. The data suggests that oxidation status of the iron in the Hb molecule and concentration of Hb play an important role in causing EC injury. The highest toxicity was observed when EC were incubated with 0.1 mM of UHb-FeIV-OH (ferryl-Hb) and no toxicity with 0.3 mM of Hb-FeIII (ferric-Hb). The direct stimulation of EC by Hb for the production of IL-1 was limited, related only to high molecular weight Hb polymers or to Hb+E, however GM-CSF expression was increased by almost all Hb forms. TNF induced dose-related injury (R2 = 0.986), and dose-related release of IL-1 (R2 = 0.977). A different EC reaction was observed on the release of GM-CSF. Intermediate levels of TNF (400 pg/mL) increased the expression of this cytokine, while high levels (800 pg/mL) blocked its release.

Reduced bovine hemoglobin solution evaluated for use as a blood gas quality-control material

We evaluated a new commercially available partially purified reduced bovine hemoglobin solution (RBHS) and a new tonometer for use in the quality control of blood gas analyzers. RBHS is manufactured in three different formulations, each corresponding to three different bicarbonate-buffering capacities and concentrations of total hemoglobin (ctHb). The P50 for each formulation of RBHS was determined to be as follows: 30.6 mmHg (4.08 kPa) for Level 1, 29.1 mmHg (3.88 kPa) for Level 2, and 28.2 mmHg (3.76 kPa) for Level 3. When RBHS and human blood samples were tonometered at three clinically significant values of PO2 and PCO2, the recovered values for PO2 and PCO2 in RBHS were comparable with those of the tonometered whole blood. Each level of RBHS also produced precise pH (SD < or = 0.006 pH units) and ctHb (SD = 1.0 g/L) values. In addition, when the temperature of a sample chamber was intentionally altered, the changes in RBHS blood gas values closely approximated the changes seen with human blood. RBHS shows more thermal sensitivity than either perfluorocarbon emulsions or aqueous buffers, which are currently being used as quality-control and proficiency testing materials.

Oxygenation of tumors by a hemoglobin solution

Tumor oxygen tensions were measured using a computer-controlled PO2 microelectrode in two preclinical solid tumor models, the rat 9L gliosarcoma and the rat 13672 mammary carcinoma. Tumor oxygenation profiles were determined under four conditions: (a) during normal air breathing, (b) during carbogen breathing, (c) after intravenous administration of a solution of ultrapurified polymerized bovine hemoglobin with normal air breathing and (d) after intravenous administration of a solution of ultrapurified polymerized bovine hemoglobin with carbogen breathing. Both tumors had severely hypoxic regions under normal air-breathing conditions. Although carbogen breathing increased the oxygenation of the better-oxygenated portions of the tumor, it made no impact on the severely hypoxic tumor regions. Administration of the hemoglobin solution was effective in increasing the oxygenation throughout both tumors under normal air-breathing conditions. The addition of carbogen breathing to administration of the hemoglobin solution eliminated severe hypoxia in the 9L gliosarcoma and markedly reduced the severely hypoxic regions of the 13672 mammary carcinoma. At 24 h after administration of the hemoglobin solution the 13672 mammary carcinoma showed greater hypoxia than before treatment, which was partially corrected with carbogen breathing.

Effect of hemoglobin solution on the response of intracranial and subcutaneous 9L tumors to antitumor alkylating agents

The 9L gliosarcoma growing subcutaneously in the hind leg of the Fisher 344 rat contains major areas of severe (< 5 mmHg) hypoxia, making up about 49% of the tumor. Intravenous administration of an ultrapurified polymerized bovine hemoglobin solution (8 ml/kg) along with normal air breathing reduces the percentage of severe hypoxia to about 24% and increases oxygenation throughout the tumor. Coadministration of the hemoglobin solution increased the tumor growth delay of subcutaneously implanted 9L tumors treated with carmustine (BCNU), cyclophosphamide, or ifosfamide but did not significantly change the tumor growth delay produced by cisplatin (CDDP). Coadministration of the hemoglobin solution with each of the four antitumor alkylating agents resulted in a near doubling of the percentage of increase in life span in animals bearing intracranial tumors treated with the combination as compared with animals treated with the drugs alone. Increases in serum blood urea nitrogen (BUN) and creatinine levels in treated animals returned to normal by 11 days posttreatment. Major changes in liver enzymes occurred with the combination of cyclophosphamide and the hemoglobin solution at 4 days posttreatment; however, these values returned to the levels in the untreated control animals within 1 week thereafter. These results indicate that further exploration of the use of hemoglobin solutions in cancer therapy is warranted.

Effect of a bovine hemoglobin preparation on the response of the FSaIIC fibrosarcoma to chemotherapeutic alkylating agents

Polymerized bovine hemoglobin solutions (PBHS) are being actively investigated as blood substitutes. In studies analogous to those we conducted with perfluorochemical emulsions/carbogen, we have examined the effect of PBHS +/- carbogen (95% O2, 5% CO2) breathing on the antitumor efficacy of melphalan, cyclophosphamide, N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) and cis-diamminedichloroplatinum(II) (cis-platin). The tumor growth delay of the FSaIIC fibrosarcoma treated with melphalan (10 mg/kg), cyclophosphamide (150 mg/kg), cisplatin (10 mg/kg) and BCNU (15 mg/kg) was increased about 2.2-fold, about 2.1-fold, about 1.2-fold and about 1.5-fold, respectively, when PBHS (12 mg/kg) was administered i.v. before each drug was injected i.p. The tumor growth delay produced by each drug was further increased when carbogen breathing for 6 h was allowed after administration of the drug and PBHS. In tumor cell survival experiments 24 h following drug treatment, the addition of PBHS increased the tumor cell killing of both melphalan and cyclophosphamide by about a factor of 10 at the lowest doses of each drug tested (10 mg/kg for melphalan and 100 mg/kg for cyclophosphamide) compared to the drug alone. However, at higher drug doses this effect was lost. The toxicity of each antitumor agent toward bone marrow (granulocyte/macrophage-colony-forming units) was increased 2- to 3-fold by the combined treatment. These results suggest that use of PBHS +/- carbogen breathing may add significantly to the efficacy of antitumor alkylating agents, however, the in vivo/in vitro data suggest that there will be increased bone marrow toxicity with this approach. This needs to be taken into account in the design of clinical trials.

Shear‐wave detection of structural effects in aqueous solutions of bovine serum albumin and hemoglobin

Using cylindrical quartz crystal torsional resonators operating at 39 and 75 kHz to generate shear waves in aqueous solutions of the proteins bovine serum albumin and hemoglobin and the polypeptide poly l-glutamic acid, it has been possible to determine the complex dynamic shear viscosities of the solutions. The effects of concentration, pH, and denaturation using various agents have been studied. It is possible to relate the viscosity and configurational elasticity of the solutions, to the intramolecular and intermolecular forces associated with the of the proteins at frequencies between 60 and 400 kHz and attributed to conformational changes of bovine serum albumin and the quaternary doublet interactions of hemoglobin have been confirmed and emphasized by the use of shear waves.

Effect of oxygen level on the enhancement of tumor response to radiation by perfluorochemical emulsions or a bovine hemoglobin preparation

Attempts to correct tumor hypoxia with oxygen-carrying solutions have used high concentrations of inspired oxygen (Fi0 2 100% or 95%). In the clinic, however, obtaining such high levels of FiO 2 using mask ventilation in older patients or in children may be difficult. Since lower levels of F102 had not been previously tested, we examined the antitumor efficacy of FiO. levels of 65, 85, and 95% breathed for 1 hr prior to and during irradiation used with the concentrated perfluorochemical emulsion F44E, the less concentrated emulsion, FluosolDA, or a new preparation consisting of purified bovine hemoglobin solution, PBHS. When tested in mice bearing the Lewis lung carcinoma with 2, 3, or 4 Gy daily for 5 days, daily Fluosol-DA produced only a small increase in the slope of the tumor growth delay versus irradiation alone, when used with 85% FiO 2 (dose modifying factor [DMF] 1.3), but produced a DMF of 2.1 with 95% FiO 2 Various concentrations of F44E (2, 4, or 8 g PFC/kg) each required a 95% FiO 2 for full effect but the 8 g/kg dose had a discernable effect with an FiO 2 of 65% and 85% (DMF 1.25 and 1.30, respectively). For PBHS, in contrast, a DMF of 1.6 was observed at 20% FiO 2 but surprisingly this increased further to 2.1 with 95% FiO 2 Further investigations of PBHS with irradiation demonstrated that daily administration of PBHS (12 ml/kg) 1 hr before single Xray fractions of 5, 10, 15, or 20 Gy with 20% FiO, resulted in a DMF of 1.6–1.7 in the FSaIIC fibrosarcoma compared with irradiation alone when ascertained by tumor cell excision assay. These results indicate that to achieve maximum antitumor benefit with these oxygen-carrying solutions with radiation therapy, care must be taken to insure that FiO 2 levels near 100% are achieved.

Hemodynamic effects and oxygen transport properties of a new blood substitute in a model of massive blood replacement

Recent concerns regarding the safety of the national blood supply have rekindled interest in the development of blood substitutes. Clinical studies have dampened the initial enthusiasm for fluorocarbon solutions as blood substitutes. The potential of hemoglobin solutions as blood substitutes has continued to stimulate investigations. However, the development of an ideal hemoglobin-derived blood substitute has eluded investigators for the past century. A persistent problem has been the inability to develop hemoglobin solutions that provide adequate oxygen and carbon dioxide exchange, while avoiding toxicity that precludes clinical safety and long-term survival. Traditionally, investigators have focused on human hemoglobin solutions. The use of outdated banked blood or pedigree human donor blood as a hemoglobin source poses continued disease transmission risks and a prohibitively limited supply. We evaluated the hemodynamic and gas transport effects of a new purified, polymerized bovine hemoglobin preparation. Bovine hemoglobin oxygen affinity is regulated by chloride ion. The concentration of chloride ions in human plasma results in excellent oxygen transport properties in a stroma-free environment. In addition, unlike human blood, bovine blood is a more disease-free hemoglobin source that is available in large supply. We exchange-transfused eight conscious sheep with this new polymerized bovine hemoglobin solution. All animals tolerated greater than or equal to 95% exchange transfusion to reach a final ovine hematocrit of 2.4 +/- 0.5% with stable hemodynamics and no clinical evidence of distress. The exchange transfusion with bovine hemoglobin polymer resulted in a final plasma hemoglobin concentration of 6.1 +/- 1.6 gm/dl, which supported oxygen consumption at baseline levels. All animals that were exchange transfused with this preparation survived long term with rapid resynthesis of ovine erythrocytes.

Preparation of microporous membranes from cation exchange membranes

A cation exchange membrane was prepared by the soaking technique. First, a base membrane was prepared by copolymerizing styrene and divinylbenzene in a poly(vinyl chloride) film and subsequently sulfonating it. The cation exchange membrane (Fe 3+ form) was then treated with an aqueous H 2O 2 solution to decompose the cation exchange resin component and was thereby transformed to a microporous membrane with no cation exchange capacity. Preparation conditions of the base membrane were studied as they relate to the characteristics of the resulting microporous membrane. Also, the suitability of the microporous membrane for ultrafiltration was studied using an aqueous solution of bovine hemoglobin.

Fractionation of oxygen isotopes due to equilibration of oxygen with hemoglobin.

The aim of the study was to answer the question as to whether the chemical reaction of oxygen with hemoglobin exhibits a source of isotopic fractionation, which could be of significance in forming the overall fractionation effect of respiration recently determined in man. Investigations were performed on bovine hemoglobin solutions adjusted to normal values of Hb concentration and pH. After degassing in vacuo, the hemoglobin solution was equilibrated in a closed system with pure oxygen of suitable pressure so that oxygen saturation levels of 30, 50 and 100% were achieved. After complete equilibration, isotope analysis of oxygen by mass spectrometry resulted in 16O18O/16O2 ratios which were 0.35 +/- 0.02% lower in the oxygen bound to hemoglobin than that of the gas phase during all levels of oxygenation. Model calculations suggest the following: (1) the fractionation during oxygen uptake in the lung at rest is primarily determined by the reaction with hemoglobin, (2) the overall fractionation effect of respiration can be explained as due to single effects of the constituent processes when assuming the oxygen transport to be limited by utilization.

Inclusion of antioxidants in resuscitation fluids.

Hemorrhagic shock is, whenever possible, treated with rapid volume replacement. Such treatment is often initiated by paramedical personnel as soon as possible following blood loss, e.g., at the site of an accident and during transport to an emergency room. The fluids used for this type of volume replacement include crystalloids, i.e., solutions of sodium chloride and other inorganic salts. Other volume expanders include biocompatible polysaccharides, so-called colloids, such as hydroxyethyl starch, dextran and human albumin. A considerable amount of research is aimed at developing resuscitation fluids based on solutions of human and bovine hemoglobin. Such stroma-free hemoglobin solutions are intended to deliver oxygen to critical organs where severe hypoxia may lead to irreversible damage.KeywordsHemorrhagic ShockIron ChelatorHydroxyethyl StarchOxygen Radical ScavengerParamedical PersonnelThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The reduction of methemoglobin levels by antioxidants.

Preventing the oxidation of hemoglobin in solution is one of the major requirements for the successful production and long-term storage of hemoglobin-based blood substitutes. To this end we have studied the effects of antioxidants on the rate of methemoglobin formation and disappearance in solutions of human and bovine hemoglobin at 4 degrees C and 37 degrees C. Ascorbate and desferal (5 mM) were observed to act as prooxidants, increasing the rate of methemoglobin formation at 37 degrees C. Trehalose, mannitol, glucose, and EDTA (5 mM) had no significant effect. Glutathione and NADH (10 mM) were the most effective antioxidants tested, causing a significant decrease in the rate of methemoglobin formation at 37 degrees C for periods of up to 50 hours. The combination of these antioxidants in bovine hemoglobin at 4 degrees C resulted in the reduction of methemoglobin levels to nearly undetectable levels in approximately 150 hours. In addition, NADH and glutathione were found to reduce methemoglobin levels to 10% over a period of 100 hours in a sample of human hemoglobin that had been stored at 4 degrees C for one year and had 60% methemoglobin. These results suggest that the prevention and reversal of methemoglobin formation during the long-term storage of hemoglobin solutions and hemoglobin-based blood substitutes may now be possible.

Aging effect of a glucose sensor immersed in aqueous glucose-bovine serum hemoglobin solutions.

A glucose sensor consisting of an enzyme membrane and an oxygen electrode was used to measure the glucose concentration in aqueous bovine serum hemoglobin solution. The enzyme membrane was an immobilized glucoxidase-PVC membrane having a finely porous structure.During application of the sensor to the solutions, bovine serum hemoglobin adsorbs on the surface of pores of the PVC membrane. Owing to the plugging of hemoglobin within the membrane, the steady-state output falls with time.Based on the reaction-diffusion model for the enzymatic reaction in the membrane, the diffusion coefficients and the reaction rate constant were evaluated. These parameters were successfully correlated with time. Calculated values of declining output of the sensor at steady state with time agreed well with the observed ones.

Evaluation of the Cd/hemoglobin affinity assay for the rapid determination of metallothionein in biologival tissues

A modification of the Cd/hemoglobin affinity assay described by S. Onosaka, T. Keiichi, M. Doi, and O. Kunio (1978, Eisei Kagaku 24, 128–131) was evaluated for its ease, sensitivity, and specificity for quantifying tissue levels of metallothionein (MT). The five-step method involves (1) preparation of the heat-denatured tissue homogenate supernatant fraction, (2) incubation of the supernatant fraction with 1 μg/ml 109Cd solution, (3) addition of 2% bovine hemoglobin solution, (4) heat precipitation of hemoglobin, and (5) analysis of the supernatant fraction for Cd remaining in the fraction. After purification of MT by gel permeation and ionexchange chromatography and analysis of each fraction for Cd-binding potential, Cd remained in the supernatant fraction only in those samples where MT characteristically elutes. The assay was capable of detecting 40 ng of MT, equivalent to a tissue concentration of 0.8 μg MT/g. The relative standard deviation for replicate assays was less than 4% of the mean. Treatment of rats with CdCl 2 demonstrated the ability of this assay to detect both large and small changes in tissue content of MT after Cd treatment in a variety of tissues. Althogh several potential sources of error are recognized, the ease and sensitivity of this assay should provide a useful means for estimating MT content in most biological tissues and should be particularly useful for large-scale studies where time and expense are major considerations.

Nonlinear acoustics and biological media

The propagation of nonlinear ultrasonic waves in aqueous solutions of biological macromolecules (tissue models) has been studied by making measurements of the fundamental at the sound source and the second harmonic as a function of distance from the source. These studies show that the nonlinearity parameter B/A for bovine serum albumin and hemoglobin solutions increases nearly linearly with concentration from 5.2 for water to 7.3 for a 40% solution, suggesting that the solution becomes more nonlinear as the spacing distance between molecules decreases. Solutions of dextran molecules also have been studied from the monomer to a molecular weight of 2 000 000 Daltons showing that the nonlinearity parameter, for fixed concentration, is relatively insensitive to molecular weight. Measurements have also been made in whole mammalian blood which exhibits a nonlinearity parameter in proportion to its dry weight which consists largely of protein. These findings suggest that details of nonlinear acoustic propagation in living systems may contribute to further improvement in clinical diagnostic procedures. [This research was supported by a grant from the National Science Foundation.]