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TOXICOLOGY

Effects of Endogenous Ascorbate on Oxidation, Oxygenation, and Toxicokinetics of Cell-Free Modified Hemoglobin after Exchange Transfusion in Rat and Guinea Pig

Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland (P.W.B., F.D., A.I.A.); and Division of Veterinary Services, Office of the Center Director, National Institutes of Health, Bethesda, Maryland (V.H.)

Received May 30, 2007; accepted July 9, 2007.

	Abstract

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Chemically modified hemoglobin (Hb) solutions are promising oxygen therapeutics; however, these agents are prone to intravascular oxidation. Using a 50% exchange transfusion (ET) model with bovine polymerized hemoglobin (PolyHbBv), we examined heme oxidation, oxygenation markers, and toxicokinetics in rats, an ascorbic acid (AA)-producing species, and in guinea pigs, a non-AA-producing species. Plasma AA decreased by 50% in guinea pigs after ET, but it was unchanged in rats for the first 20 h post-ET. Both species cleared PolyHbBv from the circulation at similar rates. However, exposure to ferric PolyHbBv over time was 5-fold greater in the guinea pig. Mass spectrometry analysis of plasma revealed oxidative modifications within the tetrameric fraction of PolyHbBv in guinea pig. Oxygen equilibrium curves of PolyHbBv measured in plasma after ET were more left-shifted in guinea pigs compared with rats, consistent with increased ferric PolyHbBv formation. Renal hypoxia-inducible factor (HIF)-1, whose activity strictly depends on the partial pressure of oxygen increased over time, and it correlated inversely with circulating ferrous PolyHbBv in both species. Interestingly, HIF-1 activity was greater in guinea pigs compared with rats at 72 h post-ET. Mean arterial pressure increases were also greater in guinea pigs; however, minimal differences in cardiac and renal pathology were observed in either species. The present findings suggest the importance of plasma AA in maintaining the stability of acellular Hb susceptible to oxidation, and they may be relevant to humans, which display a similar plasma/tissue antioxidant status to guinea pig.

HBOCs have generally demonstrated promising safety and efficacy in animals, and, in many cases, favorable phase I clinical safety (Przybelski et al., 1996; Carmichael et al., 2000). However, recent well publicized late-phase clinical trial failures with certain HBOCs suggest that preclinical animal testing may not have been sufficiently predictive of safety in humans (Sloan et al., 1999; Kim and Greenburg, 2004). This recognition has highlighted the need for improved animal models and biomarkers to better predict and monitor the safety and efficacy of HBOCs in humans. In this regard, the selection of animal species to evaluate HBOCs is critical for predicting safety in both normal and disease-state human subjects.

In the present study, we compare the rat, recognizing its popularity as a small animal species used in nonclinical HBOC studies, and the guinea pig as a potentially more relevant small animal species for predicting human safety. The rationale for evaluating the guinea pig is based on several factors that suggest a similarity with humans in terms of overall antioxidant status. Guinea pigs, like humans, are incapable of endogenous production of ascorbic acid (AA) due to the evolutionary loss of functional hepatic L-gulonolactone oxidase (LGO). In contrast, rats generate 38 µg AA/mg protein/h under normal conditions and even greater amounts when subjected to stress (Chatterjee, 1973). Interestingly, extensive investigations of several HBOCs in nonclinical studies often involving AA-producing species, such as rat and swine, have demonstrated acceptable safety profiles.

Guinea pigs and humans seem to have compensated for the lack of endogenous AA production by increasing tissue antioxidant enzyme content and efficiency. For example, copper and zinc superoxide dismutase enzymatic activity in kidney and liver is approximately 2-fold higher in humans and guinea pigs compared with rats (Nandi et al., 1997). More recently human and guinea pig but not rat red blood cells have been found to possess a similar oxidoreductase system, cytochrome b₅₆₁, responsible for efficiently recycling dehydroascorbate (DHA) to functional AA (Su et al., 2006). Therefore, the general antioxidant status of guinea pig and human are quite similar so that the balance of reductive capacity is tilted toward the tissue and away from the plasma in guinea pigs and humans. Germane to these similarities is that plasma antioxidant capacity is necessary for maintaining HBOCs in a reduced form so that they are able to carry and deliver oxygen efficiently, maintain heme stability, and limit toxicity. This is especially important when tissue antioxidant status is diminished during hemorrhagic shock and ischemic conditions.

In the current study, rats and guinea pigs were subjected to 50% exchange transfusion with bovine polymerized hemoglobin (oxyglobin; PolyHbBv), a Food and Drug Administration-approved HBOC for veterinary use. We identified a strong correlation between plasma AA levels and the susceptibility of PolyHbBv to undergo oxidation, and we correlated these events for the first time with tissue oxygen-sensing mechanisms. Despite dramatic interspecies differences in plasma PolyHbBv oxidation and stability, minimal differences were found comparing the overall effect on cardiac and renal pathology, which may reflect differences in local tissue antioxidative mechanisms. These data are consistent with the general safety of certain HBOCs in normal human subjects treated in early phase clinical trials. However, more extensive work using the guinea pig in models of tissue compromise (e.g., ischemia/reperfusion) when tissue oxidative status is reduced may ultimately provide answers to why certain HBOCs fail in pivotal clinical trials.

	Materials and Methods

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Solutions. Oxyglobin (PolyHbBv) was purchased from Biopure Corporation (Cambridge, MA). This solution consists of a heterogeneous mixture of glutaraldehyde-polymerized bovine hemoglobin at a concentration of 13 g/dl in modified lactated Ringer's. A detailed description of the physicochemical properties of the mixture as a whole and of each individual fraction has been described previously (Alayash et al., 2001; Buehler et al., 2005).

Animals and Surgical Preparation. Male Sprague-Dawley rats and Hartley guinea pigs were purchased from Charles River Laboratories, Inc. (Wilmington, MA), and they were acclimated for 1 week upon arrival to the Food and Drug Administration's Center for Biologics Evaluation and Research animal care facility (Bethesda, MD). All animals were fed normal diets throughout the acclimation period, and they weighed 350 to 450 g at the time of study. Animal protocols for each species were approved by the Food and Drug Administration's Center for Biologics Evaluation and Research Institutional Animal Care and Use Committee with all experimental procedures performed in adherence to the National Institutes of Health guidelines on the use of experimental animals (Institute of Laboratory Animal Resources, 1996).

On days of surgery, rats and guinea pigs were anesthetized via the i.p. route with a cocktail of 100 mg/kg ketamine HCl and 5 mg/kg xylazine HCl (Phoenix Scientific Inc., St. Joseph, MO). Under aseptic conditions, a midline incision was made around the neck region, allowing for blunt dissection and exposure of the right common carotid artery and the left external jugular vein. Saline-filled catheters containing 50 IU of heparin per ml prepared from sterile PE50 tubing (Clay Adams, Parsippany, NJ) were placed in each vessel, and they were tunneled under the skin to the back of the neck. Immediately following surgeries, animals were administered a subcutaneous dose of buprenorphine (0.1 mg/kg) (Reckitt and Coleman Corp., Kingston, UK), and they were allowed 24 h of recovery before experimentation. For blood pressure measurements, the right femoral artery was also catheterized with PE10 tubing fused to PE50 tubing to allow for simultaneous administration of PolyHbBv and monitoring of arterial blood pressure. The right carotid artery catheter was connected to a Gould P23 XL pressure transducer (Gould Instrument Systems Inc., Valley View, OH) for recording blood pressure. Arterial blood pressure was recorded continuously at 100 Hz using an MP100A-CE data acquisition system (Biopac Systems, Inc., Santa Barbara, CA). Data were analyzed off-line using AcqKnowledge software (Biopac Systems, Inc.) to determine mean arterial pressure (MAP) from the following formula: [diastolic + 1/3(systolic – diastolic)], with each being averaged over consecutive minutes of acquired data.

Blood/Tissue Collection. Blood (350 µl) was sampled (before infusion), immediately after infusion, and at 4, 12, 24, 48, and 72 h after end of exchange transfusion (ET) for the following analyses: 1) plasma AA, 2) hematocrit (Hct), and 3) plasma oxygen equilibrium values. Heart and kidneys were harvested at the same time points for histopathology and renal tissue hypoxia-inducible factor (HIF) activity. Each time point represents an n = 3 to 5 animals.

In a separate group of animals (n = 5–6), blood samples (0.2 ml) were obtained from the arterial catheter before infusion (baseline) and at the end of ET (time 0), and at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 8, 12, 24, 48, and 72 h after the end of ET. Plasma was used for evaluation of 1) total PolyHbBv, 2) ferrous PolyHbBv, 3) ferric PolyHbBv, and 4) PolyHbBv polymeric component distribution.

Plasma Ascorbic Acid Analysis. Plasma AA was analyzed by a modified reverse-phase high-performance liquid chromatography method. In brief, plasma samples (200 µl) were centrifuge-filtered at 12,000 rpm for 45 min at 4°C using Microcon YM-10 filter tubes (Millipore Corporation, Bedford, MA) to remove PolyHbBv and other proteins greater than 10,000 Da. Standard curves and controls were prepared in normal saline using AA (Sigma-Aldrich, St. Louis, MO) and acetaminophen (Sigma-Aldrich) as the internal standard. All standards and controls were subjected to the same filtration steps. Total AA (oxidized and reduced AA) was evaluated by adding 5 µl of a 100 mM solution of dithiothreitol (Sigma-Aldrich) to each filtered sample to reduce any DHA. Standards and samples (50 µl) were run on a PrimeSep D (4.6 x 100 mm; 5 µm) reverse-phase column with a PrimeSep D (4.6 x 10 mm) guard column (SIELC Technologies, Inc., Prospect Heights, IL) attached to a Dionex Summit P680 pump with a Dionex UVD 170S detector (Dionex Corp., Sunnyvale, CA). The mobile phase consisted of 40% acetonitrile /1% acetic acid, and it was pumped at a rate of 1 ml/min. Absorbance was measured at 254 nm for detection of AA and acetaminophen.

Pharmacokinetic/Toxicokinetic Analysis. Fully conscious and freely moving rats (n = 6) and guinea pigs (n = 5) underwent a 50% ET, replacing blood with PolyHbBv. Arterial and venous catheters were extended, tethered, and connected to separate syringe pumps (model 11; Harvard Apparatus Inc., Holliston, MA) set on withdrawal (1 ml/min) and infuse (1 ml/min), respectively. The 50% ET volume in the rat was calculated as 50% ET (milliliters) = [0.06 (milliliters per gram) x body weight (grams) + 0.77]/2 (Lee and Blaufox, 1985) and as 50% ET (milliliters) = [0.07 (milliliters per gram) x body weight (grams)]/2 in the guinea pig (Ancill, 1956). Plasma from blood in the heparinized withdrawal syringe for each transfused animal was obtained to determine the total PolyHbBv removed during the exchange transfusion period (approximately 12 min). Blood samples (0.2 ml) were obtained from the arterial catheter before infusion (baseline) and at the end of ET (time 0), and at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 8, 12, 24, 48 and 72 h after the end of ET.

Plasma concentrations of ferrous PolyHbBv and ferric PolyHbBv were determined using a photodiode array spectrophotometer (model 8453; Hewlet Packard, Palo Alto, CA). Plasma from the baseline (pre-ET) sample for each animal was used to correct for background interference and turbidity. Concentrations of ferrous PolyHbBv (oxy/deoxy), ferric PolyHbBv, and hemachrome were determined using a multicomponent analysis based the extinction coefficients for each Hb species (Winterbourn, 1985). Electron paramagnetic resonance was also used to confirm that ferric PolyHbBv in the circulation was primarily in its nonliganded state as opposed to partially liganded or fully liganded (e.g., Fe³⁺-NO) forms, which are indistinguishable by UV-visible spectrophotometry (data not shown).

Pharmacokinetic Analysis. The dose (milligrams) of PolyHbBv received by each animal at the end of ET was determined by subtracting the total amount of PolyHbBv in the plasma from whole blood collected in the ET syringe from the total amount of infused PolyHbBv according to the following equation: dose_received = ([PolyHbBv]_inf x V_inf) – ([PolyHbBv]_totalET x V_ET, where dose [PolyHbBv]_inf is the concentration of PolyHbBv (milligrams per milliliter) infused, V_inf is the PolyHbBv infusion volume (milliliters), [PolyHbBv]_totalET is the concentration of PolyHbBv (milligrams per milliliter) from plasma sampled out of the withdrawal syringe, and V_ET is the volume (milliliters) collected in the withdrawal syringe. MALDI-MS was performed to confirm that no Hb originating from red blood cells (RBCs) was present in the withdraw syringe sampled plasma. MALDI-MS was also performed on both rat and guinea pig plasma to explore stability of circulating PolyHbBv fractions as described under Results (Fig. 4).

View larger version (25K): [in this window] [in a new window]   Fig. 4. A and B, MALDI-MS evaluation of HbG globin chain destabilization in plasma sampled from rat and guinea pig. Aa, MALDI-MS spectra of rat red blood cell hemoglobin obtained from rats whole blood sampled at baseline. The [M + H]1+ ion at m/z 15201.6 and m/z 15851.51 represent the primary and globin chain ions in rat red cells and serves as a control for the presence of red cell hemoglobin, which could be present in plasma samples (a and b). Ab, MALDI-MS spectra of rat plasma sampled at 4-h postexchange transfusion. The [M + H]1+ ions denoted *at m/z 15328.46, * m/z 16110.63, and *-* m/z 30940.17, represent the tetrameric fraction of , , and - cross-linked species of PolyHbBv. Ac, MALDI-MS spectra of rat plasma sampled at 24-h postexchange transfusion. The [M + H]1+ ions denoted *at m/z 15285.00, * m/z 16108.70, and *-* m/z 30932.26 represent the tetrameric fraction of , , and - cross-linked species of PolyHbBv. The arrow indicates the [M + H]1+ ion for albumin; this ion becomes more abundant (less suppressed) as PolyHbBv is cleared from the plasma. Ba, MALDI-MS spectra of guinea pig red blood cell hemoglobin obtained from guinea pig whole blood sampled at baseline. The [M + H]1+ ions at m/z 15228.59 and m/z 15921.00 represent the primary and globin chain ions in guinea pig red cell hemoglobin and serve as a control for the presence red cell hemoglobin, which could be present in plasma samples (a and b). Bb, MALDI-MS spectra of guinea pig plasma sampled at 4-h postexchange transfusion. The [M + H]1+ ions denoted *at m/z 15160.21, *at m/z 16093.84, and *-*at m/z 31025.1 represent the tetrameric fraction of PolyHbBv , (shown in blue in the full spectra and inset) and - cross-linked species of PolyHbBv. Bc, MALDI-MS spectra of guinea pig plasma sampled at 24-h postexchange transfusion. The [M + H]1+ ions denoted *at m/z 15250.81, *at m/z 16125.87, and *-*at m/z 31009.68 represent the tetrameric fraction of , (shown in red in the full spectra and inset) and - cross-linked species of PolyHbBv. The increase in intensity of the * ion could not be assigned to the guinea pigs own red cell Hb (m/z 15228.59); thus, the intensity increase is consistent with - cross-link destabilization. The arrow indicates the [M + H]1+ ion for albumin, this ion becomes more abundant (less suppressed) as PolyHbBv is cleared from the plasma.

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Plasma Oxygen Equilibrium Curve Measurements. Oxygen equilibrium curves (OECs) were obtained using a Hemox analyzer (TCS Scientific, New Hope, PA). Experiments were carried out in 0.1 M phosphate buffer/0.1 M NaCl at pH 7.4, and the temperature was maintained at 37°C. To prevent formation of ferric PolyHbBv, 4 µl of the Hayashi enzymatic reduction system was added to the 4-ml solution (Hayashi et al., 1973). Oxygen equilibrium parameters were derived by fitting the Adair equations to each OEC by the nonlinear least-squares procedure included in the Hemox analyzer software (p50 PLUS, version 1.2; TCS Scientific) (Alayash et al., 2001). The Adair constants were then used to generate an OEC curve to generate the P₅₀ and n₅₀ (Hill coefficient) for oxygen binding. Therefore, the procedure made it possible to measure the oxygen binding parameters of PolyHbBv, which is not fully saturated at atmospheric oxygen partial pressures (Nagababu E et al., 2002).

PolyHbBv Polymer Distribution and Globin Chain Dissociation in Plasma. Plasma samples (50 µl) were evaluated by size exclusion chromatography (SEC) to compare distribution of HbG polymeric components at time points post-ET. Samples were run on a BioSep-SEC-S3000 (600 x 7.5 mm) SEC column (Phenomenex, Torrance, CA) attached to a Waters 626 pump and Waters 2487 dual-wavelength detector, controlled by a Waters 600s controller using Millenium32 software (Waters, Milford, MA). The running buffer consisted of 0.1 M NaH₂PO₄, pH 6.5, pumped at rate of 0.5 ml/min, and the absorbance was monitored at 405 nm.

Plasma samples (10 µl) were desalted using C18 ZipTips (Millipore) according to the manufacturer's instructions. A 1-µl aliquot was pipetted onto a stainless steel MALDI-MS sample plate and mixed with 1 µl of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) (Sigma-Aldrich) saturated in 50% acetonitrile/0.1% trifluoroacetic acid. The sample/matrix was air-dried and analyzed on a PerSeptive Biosystems DERP MALDI-time of flight mass spectrometer calibrated manually with purified human serum albumin (Sigma-Aldrich) and using Voyager 5.1 software with Data Explorer (Applied Biosystems, Framingham, MA) operated in linear mode.

Blood Pressure. Fully conscious and freely moving rats (n = 5–6) and guinea pigs (n = 5–6) underwent a 50% ET replacing blood with PolyHbBv (13 g/dl). Withdrawal of blood and infusion of PolyHbBv took place simultaneously at a rate of 1 ml/min from the right femoral artery and the left external jugular vein, respectively. The right carotid artery catheter was connected to a Gould P23 XL pressure transducer (Gould Instrument Systems Inc.) for recording blood pressure. Arterial blood pressure was recorded continuously at 100 Hz using an MP100A-CE data acquisition system (Biopac Systems, Inc.). Data were analyzed off-line using AcqKnowledge software (Biopac Systems, Inc.) to determine MAP from the following formula: [diastolic + 1/3(systolic – diastolic)], with each being averaged over consecutive minutes of acquired data.

Hypoxia-Inducible Factor-1 DNA Binding Assay. HIF-1 DNA binding activity of animal tissues was determined by using an enzyme-linked immunosorbent assay-based method and kit (Trans-binding HIF-1 Assay kit; Panomics, Redwood City, CA) according to manufacturer's specifications. HIF-1 transcriptional factor was assessed by detection of its binding to an oligonucleotide containing the hypoxia-responsive element (5'-TACGTGCT-3') after lysis, and nuclear extraction was achieved with the use of a nuclear extraction kit provided by the manufacturer (Panomics). HIF-1 binding was detected by a mouse antibody directed against HIF-1 region available after DNA binding and revealed by anti-mouse IgG coupled to horseradish peroxidases, which provides sensitive colorimetric detection by a spectrophotometric microplate reader at 450 nm.

Histopathology. Hearts and kidneys were fixed in 10% formalin, embedded in paraffin, and then 5-µm sections were cut and stained by standard hematoxylin and eosin procedures. Tissues were scored by a veterinary pathologist using a semiquantitative grading system as follows: 0, minimal; 1, mild; 2, moderate; and 3, severe (minimal indicates a detectable process but barely present, mild indicates small aggregates of inflammatory or necrotic cells, moderate indicates large aggregated inflammatory or necrotic cells at 20x, and severe indicates large multifocal aggregates making up greater than 50% of the tissue area).

Statistical Analysis. Arterial blood pressures, heart rate, noncompartmental analyses of PK parameter estimates, hematocrit, and plasma ascorbate concentrations are expressed as means ± S.E.M. Statistical comparison for arterial blood pressures, heart rate, PK parameter estimates, hematocrit, and plasma AA concentrations were performed by analysis of variance with an a priori test for planned comparisons between the rat and the guinea pig. In all analyses, a value p < 0.05 was taken as the level of statistical significance.

	Results

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Plasma AA Levels following Exchange Transfusion. Plasma reductants such as AA have been shown to effectively reduce oxidized Hb in humans (Ames et al., 1981). Figure 1A shows plasma AA concentrations in rats and guinea pigs following 50% ET. Baseline (BL) plasma AA concentrations in the rat and guinea pig were 100.03 ± 16.2 and 51 ± 15.8 µM, respectively. When subjected to 50% ET, rats exhibited plasma AA concentrations similar to BL at 4 h (97.2 ± 5.2 µM), 12 h (104.6 ± 7.0 µM), and 24 h (109.4 ± 21 µM) post-ET. At 48 h post-ET, plasma AA concentration decreased below BL (i.e., 51.6 ± 13 µM), and this AA concentration was maintained at 72 h post-ET (50.8 ± 17 µM). An opposite situation occurred in guinea pigs following 50% ET such that AA plasma concentrations were approximately 50% BL at 4 h (22 ± 5.7 µM), 30% BL at 12 h (15.9 ± 3.4 µM), 36% BL at 24 h (18.5 ± 5.8 µM), 41% BL at 48 h (20.9 ± 3.1 µM), and 96% BL at 72 h (48.1 ± 16 µM) post-ET. These data suggest that the rat rapidly up-regulates AA hepatic production to make up for losses during ET, whereas in the guinea pig, a 50% loss of AA plasma concentration is observed 4 h after 50% ET, and it remained low, which is consistent with a 50% exchange transfusion in a species incapable of endogenous AA production.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Plasma ascorbic acid and hematocrit in rat and guinea pig following PolyHbBv exchange transfusion. A, Pre and postexchange transfusion plasma ascorbic acid concentrations in the rat () and guinea pig (). B, pre and postexchange transfusion hematocrit in the rat (filled bars) and the guinea pig (open bars). Values are reported as micromolar ± S.E.M. Significant differences (p < 0.05) between rat and guinea pig are denoted by , whereas significant differences from baseline values are denoted by * (n = 5–6 animals/species/time point).

Efficient AA functioning is dependent on the presence of normal circulating RBCs that chemically recycle DHA back to functional AA (May et al., 2004). Given this intimate relationship between AA and RBCs, Hct levels in the two species were also measured (Fig. 1B). Baseline Hct levels were 44.4 ± 1.5 and 37.8 ± 0.85% in the rat and guinea pig, respectively, whereas ET reduced Hct levels to 21.9 ± 0.92 and 18.3 ± 0.47 in each species. Over the 72 h of evaluation, neither species demonstrated changes in Hct from end ET levels. These results indicate that the exchange transfusion resulted in similar reductions in red cell volume in both species; thus, red cell volume cannot account for the interspecies differences in plasma AA.

Ferric PolyHbBv Formation following Exchange Transfusion. To examine the possible correlation among plasma AA levels, oxidation, and oxidative stability of PolyHbBv, we measured the postexchange levels of ferric PolyHbBv (Fig. 2). The C_max for ferric PolyHbBv occurred at time 0 (end of ET) in the rat and at 12 h post-ET in the guinea pig, accounting for 2.2 and 23.2% of the total PolyHbBv concentration in each species at each T_max, respectively. The AUC_0– for ferric PolyHbBv in the rat and guinea pig was 6.6 and 34.5% of the total PolyHbBv AUC_0–, respectively. Plasma CL of ferrous PolyHbBv occurred faster in the guinea pig compared with the rat; however, no such difference was found in total PolyHbBv plasma CL.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Log-linear plasma concentration versus time profiles of PolyHbBv in rats and guinea pigs. Plasma concentrations of total PolyHbBv (), ferrous PolyHbBv (), and percentage of plasma ferric PolyHbBv () at time points from the end of exchange transfusion until 72 h in the rat (A) and guinea pig (B). Convergence of dotted and dashed lines indicates the period of greatest ferric PolyHbBv exposure. The pharmacokinetic estimates derived from these data are shown in Table 1.

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Plasma PolyHbBv Component Plasma Distribution/Globin Chain Dissociation. The SEC chromatographs of plasma from rat and guinea pig obtained from blood samples at the end of ET until 72 h post-ET were separated on a BioSep-SEC-S3000 (600 x 7.5 mm) SEC column (Phenomenex) are shown in Fig. 3, A and B. As expected the chromatograms show a more rapid loss of tetramer in each species and a slower disappearance of multitetrameric PolyHbBv components. PolyHbBv tetramer and multitetrameric species seem to be similarly eliminated from the plasma at time points post-ET. Alternatively, larger PolyHbBv multitetramers may break down to smaller mol. wt. components before elimination. However, upon closer inspection of PolyHbBv elimination curves and SEC chromatograms obtained from guinea pig plasma, both PolyHbBv tetrameric and multitetrameric fractions seem to be eliminated more rapidly in the first 4 h post-ET. This observation may be due to the accelerated oxidation of PolyHbBv in guinea pigs; thus, increased elimination may serve as an early protective mechanism removing oxidized PolyHbBv.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Size-exclusion chromatography patterns of plasma PolyHbBv over time in the rat and guinea pig. A, representative (single animal) pattern of tetrameric and multitetrameric fractions in PolyHbBv as they exist in the plasma of the rat over time. B, representative (single animal) pattern of tetrameric and multitetrameric fractions in PolyHbBv as they exist in the plasma of the guinea pig over time. In each case, plasma samples (50 µl) were run on BioSep-SEC-S3000 (600 x 7.5 mm) SEC column. The running buffer consisted of 0.1 M NaH2PO4, pH 6.5, pumped at rate of 0.5 ml/min and the absorbance was monitored at 405 nm.

In Fig. 4B, the MALDI-MS spectra of guinea pig RBC Hb demonstrates an globin chain [M + H]¹⁺ ion at m/z 15228.59 and globin chain [M + H]¹⁺ ion at m/z 15921.00. The and globin chain ions are consistent with those reported in the literature (Day et al., 1996). At 4 and 24 h post-ET, the cross-linked PolyHbBv-derived globin chains are seen as an *-*[M + H]¹⁺ ion at m/z 31025.19, whereas residual *[M + H]¹⁺ (limited percentage of intensity) and intense *[M + H]¹⁺ globin chain ions are observed at m/z 15250.81 and 16125.87, respectively. The *[M + H]¹⁺ ion at m/z 15250.81 in the spectra of the 24-h plasma sample indicates increased peak intensity of 35% from the 4-h sample, suggesting globin chain cross-link destabilization in vivo. Rat and guinea pig plasma samples did not demonstrate mass ions for or globin chains of Hb originating from rats' or guinea pigs' own RBCs. Thus, destabilized globin could only have come from PolyHbBv.

Blood Pressure and Heart Rate Response to PolyHbBv. HBOC-mediated blood pressure elevation is a common finding in animals and humans, and it can result in reduced tissue oxygenation. To investigate the possible relationship between PolyHbBv oxidative stability and the susceptibility and/or extent of hemodynamic alterations, we monitored blood pressure and heart rate in fully conscious and freely moving rats and guinea pigs (n = 5 for both species) during the course of the 50% ET. Figure 5A shows the changes in MAP between rat and guinea pig. Baseline blood pressure values monitored over 20 min in rats ranged as follows: systolic (140.4 ± 6.44–143.8 ± 6.40 mm Hg), diastolic (109.6 ± 4.93–125.2 ± 6.22 mm Hg), MAP (119.0 ± 5.81–125.2 ± 6.22 mm Hg), and pulse pressure (23.8 ± 4.41–27.4 ± 2.11 mm Hg). In guinea pigs, baseline blood pressure values ranged as follows: systolic (73.4 ± 3.60–83.2 ± 5.35 mm Hg), diastolic (48.8 ± 4.85–56.2 ± 5.76 mm Hg), MAP (60.2 ± 4.14–63.0 ± 4.10 mm Hg), and pulse pressure (23.8 ± 4.41–27.4 ± 2.11 mm Hg). Immediately after the start of ET, a concomitant drop in heart rate occurred (Fig. 5B).

View larger version (32K): [in this window] [in a new window]   Fig. 5. Cardiovascular effects of PolyHbBv in the rat and guinea pig. A, percentage of change from baseline in MAP in the guinea pig () and rat (). MAP increase compared with baseline was 20% in rats compared with 50% in guinea pigs. Values represent mean ± S.E.M. B, percentage of change from baseline in heart rate response in rat () and guinea pig (). Measurements were recorded continuously and plotted at 2-min intervals. Significant differences in MAP (p < 0.05) were observed in both the rat and guinea pig over the entire 2-h period post-transfusion compared with baseline (n = 5 for each species).

Plasma Oxygen Equilibrium Curve Analysis. The impact of increased plasma PolyHbBv oxidation on PolyHbBv oxygen-carrying capacity in both species was examined. OECs in plasma samples obtained over the course of 72 h are shown in Fig. 6, A and B, for rat and guinea pig, respectively. From plasma OECs, we were able to calculate a P₅₀ = 36.4 ± 0.989 mm Hg immediately after the end of ET in the guinea pig, compared with 41.7 ± 0.73 mm Hg in the rat. At 48 h post-ET, a greater shift toward increased oxygen affinity (reduced oxygen off-loading) was observed in the guinea pig (P₅₀ = 25.1 ± 0.441 mm Hg) compared with the rat (38.9 ± 2.72 mm Hg). These observations are consistent with the increased ferric PolyHbBv accumulation over time in guinea pigs.

View larger version (16K): [in this window] [in a new window]   Fig. 6. OECs for PolyHbBv in rat and guinea pig plasma. OECs for samples obtained post-transfusion from plasma of rats (A) and guinea pigs (B) at specific time intervals are shown. OECs were determined by automated Hemox analyzer at 37°C. All samples contained approximately 60 µM heme in a 0.01 M phosphate buffer containing 0.1 M NaCl, pH 7.4. Antifoaming agent and Hayashi reduction reagents (see Materials and Methods) were added to the buffer. Results are expressed as percentages of oxygen saturation versus partial pressure of oxygen. P50 values were obtained from these curves after correction for the incomplete saturation OECs at 100 mm Hg (see Materials and Methods).

View larger version (22K): [in this window] [in a new window]   Fig. 7. Correlation between the levels of PolyHbBv (oxy) and HIF-1 DNA binding activity in kidneys from rats and guinea pigs. Ferrous form of PolyHbBv () measured in plasma samples taken from rats (A) and guinea pigs (B) plotted as a function of HIF-1 activity as measured by enzyme-linked immunosorbent assay-based method (see Materials and Methods) and time (hours) post-transfusion with PolyHbBv.

Heart and Kidney Histopathology. To investigate whether the interspecies differences in PolyHbBv oxidation/oxygenation translated into differences in organ pathology, we performed hematoxylin and eosin staining and histopathological analysis of heart (Fig. 8) and kidneys (Fig. 9) obtained at baseline (24 h after surgery) and 24 and 72 h post-ET. Tissues from each species (n = 3–5/treatment per time point) were scored by a veterinary pathologist using a semiquantitative grading system such that mean ± S.E.M. severity scores provide a general evaluation of tissue inflammation and necrosis. Rat and guinea pig heart (left ventricle) and kidneys exhibited minimal (0) to mild (1) pathological response to PolyHbBv at 24 h post-ET in rat and guinea pig heart and kidneys with regard to inflammation and necrosis. Figure 8, B and D, shows mild and focal myofiber necrosis in the left ventricle and focal inflammation (inset). Myofiber involvement tended to resolve in both species within 72 h. Figure 9, B and D, shows glomeruli in kidneys harvested 24 h after dosing revealing no glomerular injury in either species. This was also observed in the 72-h group post-ET harvested kidney tissue. No abnormal histopathology was noted in any of the heart or kidneys tissues of animals subjected to ET with albumin (data not shown).

View larger version (118K): [in this window] [in a new window]   Fig. 8. Heart histopathological evaluation of rat and guinea pig tissue after PolyHbBv exchange transfusion. Hematoxylin and eosin-stained heart harvested at baseline (24 h after surgery) and 24 h postexchange with PolyHbBv in rat (A and B, respectively) and guinea pig (C and D, respectively). The black arrows indicate areas of pathology. In the rat heart (B), single necrotic myofibers are observed in the left ventricle. B, inset shows a small inflammatory lesion in the wall of the left ventricle. In the guinea pig heart (D), areas of necrotic myofibers are seen in the apical region of the left ventricle. These findings were mild and in some cases not observed in animals. Resolution was generally achieved by 72 h postexchange.

View larger version (131K): [in this window] [in a new window]   Fig. 9. Kidney histopathological evaluation of rat and guinea pig tissue after PolyHbBv exchange transfusion. Hematoxylin and eosin-stained kidney harvested at baseline (24 h after surgery) and 24 h postexchange with PolyHbBv in rat (A and B, respectively) and guinea pig (C and D, respectively) demonstrate no glomerular damage in either the rat or the guinea pig.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Infusion of HBOCs can lead to deleterious effects due to the spontaneous and uncontrolled oxidation of its heme iron. This process can be enhanced by endogenous oxidants, such as H₂O₂ and NO and/or their metabolites in circulation. The rapid conversion of the ferrous (oxygen carrying iron) to the ferric (nonoxygen carrying iron) may compromise the effectiveness and safety of HBOC for at least two reasons: 1) ferric Hb does not bind oxygen, and its accumulation can reduce the oxygen-carrying capacity of HBOCs; and 2) ferric Hb in the presence of oxidants can promote and sustain a vicious oxidative cycle that ultimately leads to the loss of highly cytotoxic heme. We previously reported that endogenous and/or exogenous reducing agents such as AA (Dunne et al., 2006) or selenium (Baldwin et al., 2004) can control Hb oxidation reactions in vitro and in vivo and that these reductants can potentially suppress the untoward consequences of these reactions. In the current work, we tested the hypothesis that oxidation of polymerized bovine Hb would be modulated differentially in the circulations of two species with different reductive capabilities, i.e., with and without the ability to synthesize AA, because these differences may affect Hb oxygen-carrying capabilities.

Plasma antioxidative status was clearly compromised in guinea pigs following ET as evidenced by the 50% reduction in plasma AA. In contrast, rat plasma AA levels remained slightly above baseline levels until PolyHbBv was cleared from the circulation of the animal. This probably reflects the ability of rats to generate sufficient AA to compensate for the loss of AA incurred from the ET and from its use during the reduction of PolyHbBv. In addition to AA, urate has been shown to play a significant role in maintaining the ferrous state of Hb in plasma and RBCs (Ames et al., 1981). However, both rat and guinea pig metabolize urate similarly, and they maintain mean plasma levels of 9.3 and 7.0 µg/ml, respectively (Mudge et al., 1968; Habu et al., 2003). Therefore, the influence of urate in these studies is probably not significant. Moreover, we recently estimated that in rabbits, which have approximate plasma concentrations of 50 µMAA and 30 µM urate, respectively, AA was kinetically more competent in reducing the oxidized forms (ferric/ferryl) of the infused cross-linked Hb than urate (Dunne et al., 2006).

When taking the oxidative status of PolyHbBv heme iron into account, a disconnect in the pharmacokinetics of ferric and ferrous PolyHbBv becomes apparent in the guinea pig. The in vivo oxidation of PolyHbBv in the guinea pig resulted in a 5-fold higher overall exposure (AUC_0–) to ferric PolyHbBv. Furthermore, in the guinea pig, approximately 20 and 40% of the circulating PolyHbBv was oxidized to ferric PolyHbBv by 4 and 36 h post-ET, respectively. These values correspond to earlier in vitro autoxidation experiments carried out on PolyHbBv in the absence of added antioxidants (Alayash et al., 2001). Conversely, in the rat, ferric PolyHbBv did not increase until plasma concentrations of PolyHbBv were 20-fold less than the C_max. Thus, increased levels of ferric PolyHbBv at 48 and 72 h occur at low total PolyHbBv concentrations. Our data are in agreement with several studies using large-volume HBOC administration that documented significant ferric Hb formation in sheep (Lee et al., 1995), rabbits (Dunne et al., 2006), and humans (O'Hara et al., 2001). In addition, administration of various percentages of oxidized PEGylated-Hb to rats as a 30% ET demonstrated that excess of 10% PEGylated-Hb in the oxidized form (ferric PEGylated-Hb) reduced both kidney and liver oxygen tension (Linberg et al., 1998). These observations suggest a potential for increased toxicity as a result of diminished oxygen delivery and oxidative side reactions caused by excessive ferric HBOC exposure.

Using MALDI-MS, the current study evaluated the in vivo oxidative modification as reflected in the structural stability of the tetrameric component of PolyHbBv, which constitutes 37% in the preinfused final polymer mixture. The mass spectra from rat and guinea pig plasma at 4 and 24 h post-ET demonstrated stability of the PolyHbBv tetramer in plasma sampled from rats at both early and later time points. However, PolyHbBv tetramer instability occurred in guinea pigs at later sampling time points. This observation is based on the detection of PolyHbBv derived and globin chain mass ions in plasma collected from guinea pigs. These ions could not be assigned to RBC derived Hb and globin chains. This suggests that PolyHbBv globin chains sustained oxidative changes, but they probably do not dissociate in the guinea pig. This may be a result of oxidative stress imparted on the protein, because tetramer destabilization does not occur in PolyHbBv before infusion based on previous extensive mass spectrometry analysis (Buehler et al., 2005). Conversely, rat Hb (Fig. 4Aa), guinea pig Hb (Fig. 4Ba), human HbA₀, and non cross-linked PEGylated Hb have been shown to readily dissociate in vitro (Iafelice et al., 2007). The overall impact of Hb monomer formation in vivo could result in acute renal failure based on globin chain deposition in the glomeruli of the kidneys (Savitsky et al., 1978).

In spite of some of the reported functional limitations of PolyHbBv, such as incomplete saturation of its OEC at high partial pressure of oxygen (i.e., 100 mm Hg) (Fig. 6), insensitivity to some allosteric modifiers of Hb function, and reduced cooperativity (Alayash et al., 2001), we show in this study that this Hb retained its oxygen-carrying capabilities, particularly when the oxidation of its iron was controlled, such as the case in the rat presumably by endogenous AA. Conversely, uncontrolled oxidation of this Hb in the guinea pig, as evidenced by increased levels of its ferric form and left-shifted OECs, may have compromised its ability to carry oxygen. Whether the dramatic interspecies differences in the oxidation/oxygenation profiles documented herein translate into different tissue oxygenation is not yet known. However, analysis of HIF-1 activity in kidney tissues obtained from the rats and guinea pigs may offer some clues. HIF-1, a major molecular transducer of hypoxia, is normally degraded in the presence of oxygen, a substrate for prolyl hydroxylase that targets HIF-1 to ubiquitination and destruction by the proteasome. By contrast, HIF-1 is stabilized during hypoxia, enabling control of the expression of hundreds of genes. We show for the first time a clear correlation between oxygenation state of Hb during the course of transfusion with renal HIF-1 activity in both species. Moreover, the decrease in oxygen-releasing capacity of PolyHbBv in the guinea pig due to its enhanced oxidation probably contributes to the increase in HIF-1 activity observed after 50 h post-ET. We are currently assessing HIF-1 protein levels and the expression of several HIF-1 target genes such as erythropoietin, vascular endothelial growth factor, and heme oxygenase in kidney, heart, and brain of these animals to better understand the interplay between Hb oxygenation, redox reactions, and overall oxygen hemostasis.

The MAP response in both rats and guinea pigs was similar in that elevation occurred immediately after the start of ET. However, the two species differed dramatically in the extent of MAP elevation over baseline values. It is tempting to speculate that AA in rats may have also played a part in controlling Hb reaction with NO, a critical molecule in blood pressure control. In addition to the well established role of AA in reducing Hb oxidation, AA may also contribute to Hb via other important reactions, i.e., nitrosative reactions. There has been in recent years a growing interest in endogenous NO storage compounds and the role of AA in inducing NO release from these compounds, particularly low-molecular-weight S-nitrosothiols, such as S-nitrosothiol glutathione (Foster and Stamler, 2004), or S-nitrosated proteins, such albumin (Grandley et al., 2005). Recent mechanistic studies, using an N-nitrosated tryptophan derivative, showed that the primary oxidized product of AA, DHA, efficiently consumes NO and subsequent release of ascorbyl radical (Kytzia et al., 2006). Along with oxidative status of the circulating HBOC, arterial blood pressure response can contribute to decreased efficacy and possible toxicity via reduced blood flow to vital tissues, increased workload on the heart, and acute renal injury.

Interestingly, neither the enhanced oxidation nor exaggerated blood pressure responses observed in guinea pigs compared with rats translated into increased tissue pathology. Both rats and guinea pigs demonstrated minimal histopathology in heart and kidney at 24 h post-ET, and most findings were resolved by 72 h post-ET. In both species, myocardial toxicity was limited to single myofiber necrosis of the left ventricle at 24 h. Although the absence of histopathological differences between rat and guinea pig was a surprising finding, it is likely that these observations may in fact provide useful information relative to predicting human safety of HBOCs based on animal data. In addition, this work supports the idea of early biomarker identification detectable in the absence of histopathological evaluation. Even though guinea pigs were unable to control the oxidation of PolyHbBv in the circulation, they, like humans, have increased tissue antioxidant mechanisms (Nandi et al., 1997), which may have prevented tissue injury in these otherwise normal transfused animals. Once protective antioxidant mechanisms in the tissue are overcome by events such as ischemia followed by reperfusion leading to H₂O₂ and neutrophil released hypochlorous acid accumulation, the combination of an oxidized HBOC and tissue oxidant accumulation may become overwhelming.

In summary, we show that rats, unlike guinea pigs, controlled oxidation of the infused cell-free Hb after 50% ET and that they maintained adequate plasma oxygenation with little or no change in the P₅₀ of Hb, due in large part to the presence of adequate circulating AA levels, during the first 20 h post-ET. By contrast, in guinea pigs, Hb oxidation preceded in the plasma at much higher rates probably due to the lack of endogenous sources of AA. The decreased ability of this Hb to carry oxygen due to the increased oxidation of its iron was consistent with the increased HIF-1 activity, particularly at later times post-ET. Therefore, this study has the following implications for the use of HBOCs in clinical development: 1) the guinea pig may represent an appropriate species for the early evaluation of HBOC susceptibility to oxidation and destabilization in circulation, and it may be more relevant to simulate the use of HBOCs in clinical settings associated with antioxidant depletion, such as trauma and diabetes; 2) endogenous antioxidant mechanisms in plasma, RBCs, and tissue should be taken into account when designing animal studies aimed at understanding HBOC oxygenation/oxidation reactions; and 3) coadministration of reducing agents, such as AA, may offer a simple strategy to control Hb oxidative side reactions.

	Acknowledgements

 
We thank Dr. Paul Yeh for performing DNA trans-binding assessment of HIF-1 and Francine Wood for performing oxygen equilibrium measurements.

	Footnotes

 
The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration, and they should not be construed to represent any agency determination or policy. This work was funded by a Center for Biologics Evaluation and Research/Food and Drug Administration Unmet Needs Award.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.126409.

ABBREVIATIONS: HBOC, hemoglobin-based oxygen carrier; AA, ascorbic acid; LGO, L-gulonolactone oxidase; DHA, dehydroascorbate; PolyHbBv, bovine polymerized hemoglobin; PE, polyethylene; MAP, mean arterial pressure; ET, exchange transfusion; Hct, hematocrit; HIF, hypoxia-inducible factor; MALDI-MS, matrix-assisted laser desorption ionization/mass spectrometry; RBC, red blood cell; PK, pharmacokinetic; AUC, area under the plasma-concentration time curve; CL, plasma clearance; MRT, mean residence time; V_ss, apparent volume of distribution at steady state; OEC, oxygen equilibrium curve; C_last, last measurable concentration; SEC, size exclusion chromatography; BL, baseline; PEG, polyethylene glycol.

Address correspondence to: Dr. Paul W. Buehler, Center for Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bldg. 29, Rm. 129, Bethesda, MD 20892. E-mail: paul.buehler{at}fda.hhs.gov

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