Male Sprague-Dawley rats (280–350 g and 10–12 wk of age) were obtained from a commercial vendor (Charles River, Wilmington, MA) and housed in the University of Colorado Health Sciences Center's Center for Laboratory Animal Care (elevation, 1,500 m). Animals were allowed ad libitum access to food and water, and kept on a 12-hour day–night cycle. All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at University of Colorado Health Sciences Center.
Rats were allowed to acclimate to Denver altitude (1,500 m; barometric pressure [PB] ~ 630 mm Hg) for at least 7 days before instrumentation. At 48 hours before surgery, rats were provided water supplemented with acetaminophen/codeine (0.5 mg/ml and 0.05 mg/ml, respectively) for postoperative analgesia. The animals were weighed and hematocrits were determined. Rats were anesthetized with a mixture of ketamine:Rompon (xylazine) (75:6 mg/kg, intraperitoneal). Under aseptic conditions, the left carotid artery was cannulated with a PE-50 (0.58-mm inside diameter [ID]; Becton Dickinson, Franklin Lakes, NJ) catheter. A PV-1 (0.28-mm ID; Becton Dickinson) catheter with a shallow bend at its tip was inserted into the right ventricle via the right jugular vein, and guided into the main pulmonary artery. Pressure tracings confirmed placement in the pulmonary artery. Next, two PE-50 (0.58-mm ID; Becton Dickinson) catheters were placed in the superior vena cava via the right jugular vein for venous blood collection, and to obtain cardiac output values. All catheters were flushed with heparinized saline, tied off, tunneled subcutaneously to the dorsal neck region, and exteriorized at the back of the neck. Animals were allowed at least 48 hours to recover before any treatments. Animals demonstrating signs of infection, diarrhea, or distress were excluded from study.
S-Nitrosylation of Polymerized Glutaraldehyde Hb (SNO + HBOC)
PBvHb (oxyglobin; Biopure, Cambridge, MA) is the product of a reaction with bovine Hb and glutaraldehyde performed under deoxygenation, and is thus partially locked in a T-state conformation (24
). Bovine Hb only contains two Cys residues, which are located in the highly conserved β-globin chain 93 positions, and we have previously shown that, unlike other chemically modified Hbs, this Cys remains unmodified and free for S-nitrosylation (25
PBvHb was SNO, as previously described (18
). Briefly, equal volumes of 0.5 M reduced glutathione/100 uM diethylene triamine pentaacetic acid (DTPA) and 0.5 M NaNO2/100 uM DTPA were reacted to yield SNO glutathione (GSNO). The pH of the PBvHb solution was adjusted to pH 9.2 using a KH2
buffer system. Then, a 10-fold molar excess of GSNO was reacted with the basic PBvHb in a dark reaction vessel at 4°C for 30 minutes. The reaction solution was dialyzed overnight against lactated Ringer's solution (R) to terminate the reaction and purify the sample.
S-Nitrosylation of β93 Cysteine: Confirmation, NO Quantification, and Dose
Mass spectrometry (MS) (matrix-assisted laser desorption/ionization-MS and liquid chromatography–MS) was performed to: (1
) confirm the presence of NO on the β chain of the PBvHb (2
); determine if the S-nitrosylation process may have weakened or damaged the PBvHb structure before infusion; and (3
) determine if the S-nitrosylation process altered in vivo
stability of PBvHb in rat plasma samples (). For a visual representation of SNO bond, the reader can refer to Reference 22 and . MS was performed on PBvHb from starting material and in plasma samples as previously described (24
). The data are consistent with the addition of NO to PBvHb, as well as unaltered pre- and postinfusion stability of SNO-PBvHb. An additional globin chain ion, denoted α-globin (m/z
at ~15,260 [M+H]), is apparent in both PBvHb and SNO-PBvHb end-treatment plasma samples. This ion was determined by liquid chromatography/MS/MS to be of rat red cell Hb origin (data not shown), and not consistent with the bovine Hb amino acid sequence.
Figure 1. Matrix-assisted laser desorption/ionization–mass spectrometry (MS) of polymerized bovine hemoglobin (PBvHb) and S-nitrosylated (SNO) PBvHb in vitro and in vivo are shown as (A) prenitrosylation PBvHb, (B) postnitrosylation PBvHb (SNO-PBvHb), ( (more ...)
Figure 2. End-study plasma Hb content represented as heme concentration (μM). (A) The 2-hour postinfusion ferrous and ferric content in plasma. (B) Black lines are spectra of three samples from PBvHb infused animals gray lines are spectra from Ringer's (more ...)
The Saville reaction was used to quantify the molar amount of NO bound to PBvHb, as previously described (27
) The amount of S-nitrosothiol was quantified as the difference in absorbance between samples containing solution C (1% sulfanilamide and 0.2% HgCl2
in 0.5 M HCl) and solution B (1% sulfanilamide in 0.5 M HCl). Standard curves were calculated from known concentrations of S-nitrosoglutathione stock solutions plotted against absorbance at 540 nm.
A whole-blood concentration of 1 μM SNO cell-free Hb has been shown to elicit a vasodilatory response under acute hypoxic conditions (22
). We confirmed this observation by infusion of SNO-PBvHb to achieve a final whole-blood concentration of roughly 2uM SNO-PBvHb (shown in figures, tables, and in the Results
). For all studies, SNO-PBvHb (~0.3 ml) was added to the lactated R or PBvHb solution (2.7 ml, ~1:10 mixture) immediately before a study began and infused as a total 3 ml bolus.
Stopped-Flow Kinetic Studies
Reactions of ferric Hbs with NO were measured in the stopped flow, as previously described (28
). Hb solutions (1 μM in heme) were mixed with increasing concentration of NO (up to 100 μM) to start the reaction, and the absorbance changes were monitored at 420 nm. Multiple kinetic traces were averaged, and nonlinear least-squares curves fitted to exponential equations to obtain reaction rate constants.
Oxygen Equilibrium Studies
Oxygen binding studies for Hb SNO-PBvHb were performed in a Hemox Analyzer (TCS Scientific, New Hope, PA). The samples were equilibrated with pure nitrogen gas and reoxygenated with air. The oxygen tension was measured using a Clark oxygen electrode (Model 5331 oxygen probe; Yellow Springs Instruments, Yellow Springs, OH). The oxygen saturation of Hb was monitored via a built-in, dual-wavelength spectrophotometer. A typical experiment was conducted with an Hb concentration between 60 and 75 μM (heme) at 37°C, and each experiment was repeated three times. The final solution (4 ml) contained 4 μL of the Hayashi enzymatic reduction system to maintain the metHb content to a minimum level (29
). Oxygen equilibrium curves were obtained and analyzed yielding p50 (the partial pressure of oxygen at which Hb is 50% saturated), and n50, the Hill coefficient for oxygen binding. The data analysis was performed by nonlinear least-squares curve fitting of the Adair equations derived from the Hemox Analyzer software P50 Plus version 1.2.
Model and Experimental Design
Before either normoxic or acute hypoxic exposure (10% O2, 4 h), animals were randomized into one of four groups, each with a sample size of 8–10 animals (n = 8–10): (1) lactated R (Henery Schein, Melville, NY) infused; (2) PBvHb; (3) SNO-PBvHb; and (4) SNO-PBvHB plus PBvHb. Additionally, as a comparison to the hypoxia-exposed SNO-PBvHb–treated animals, a separate group of animals (n = 3) received an infusion of GSNO (1 mM; 3 ml bolus).
Rats were placed in custom-designed, small, rectangular, Plexiglas chambers with a portal through which catheters could be passed. Catheters were flushed with heparinized saline and then connected to fluid-filled pressure transducers. All animals were exposed to hypoxia by flushing the chamber with 10% O2 gas (fraction of inspired oxygen = 10% O2). Once breathing hypoxic gas, hypoxic animals were not re-exposed to room air. Blood pressures, pulmonary artery blood pressures, heart rates, cardiac outputs, and blood gases were collected after 30 minutes of hypoxic exposure, before any treatment or infusion.
All animals then underwent a 30-minute (10 min/ml) infusion of lactated R (3 ml), PBvHb (1.3 g/kg in a 3 ml vol), SNO (2 μM whole-blood concentration; 0.3 ml SNO:2.7 ml lactated R), or SNO plus PBvHb (2 μM whole-blood concentration; 0.3 ml SNO:2.7 ml PBvHb) through a venous catheter. Hemodynamic variables were measured in all groups at 60 and 120 minutes after infusion. Blood gases were measured at baseline and 120 minutes. Cardiac output was measured using Cardiogreen (catalog no. I2633; Sigma-Aldrich, St. Louis, MO) with the dye-dilution method. Between data collection points, the rats were monitored for any signs of distress, but were otherwise left undisturbed. Animals were killed with an overdose of sodium pentobarbital (100 mg/kg) via a jugular catheter after final measurements.
Measurement of Plasma Ferrous and Ferric Hb
Plasma from the end-of-study blood samples were used to determine content of ferrous PBvHb (Fe2+, oxy/deoxy) and ferric PBvHb (Fe3+, deoxy) using a photodiode array spectrophotometer (Model 8453; Hewlet Packard, Palo Alto, CA). Blank rat plasma was used to correct for background interference and turbidity. Molar concentrations of ferrous and ferric heme in PBvHb were determined using a multicomponent analysis based on the extinction coefficients for each Hb species.
Measurement of SNO-PBbHb Solution and Plasma Nitrite Levels
Nitrite levels were determined in the SNO-PBvHb starting solution and in the end-point plasma samples collected from six (n = 6) animals in each treatment group, unless otherwise indicated, using a commercial nitrate/nitrite colorimetric assay kit (Cayman Chemical Co., Ann Arbor, MI). The assay was performed per manufacturer's instructions. Plasma samples were evaluated in duplicate using a dilution, as recommended by the manufacturer for analysis of plasma samples, whereas the noninfused SNO-PBvHb was evaluated without dilution.
Blood Gas and Co-Oximetry Measurements in Whole Blood and Plasma
Blood samples were collected immediately after hemodynamic measurements had been obtained at baseline and 120 minutes. Arterial (0.2 ml) and venous (0.2 ml) blood was withdrawn via carotid and venous catheters, respectively, into blood gas syringes, and analyzed (ABL5 and co-oximetry via OSM3; Radiometer, Copenhagen, Denmark) with algorithms specific to rat and bovine Hb.
Heparinized microhematocrit capillary tubes (100 μl, catalog no. 22362566; Fisherbrand, Pittsburgh, PA) were immediately filled from each 1-ml blood gas syringe (see above), sealed from room air with a removable cap (catalog no. 8889212000; Kendall Healthcare, Mansfield, MA), and spun on a capillary centrifuge to separate cell and plasma fractions. The plasma portion of the capillary tube was aspirated into the co-oximeter. Measurements were excluded if any portion of the cell fraction was inadvertently aspirated into the analyzer. Care was taken to avoid exposing blood samples to room air.
Calculation for Oxygen Delivery
Oxygen delivery was calculated for Hb in whole blood and PBvHb in the plasma phase (Eq.1 below). No measurable endogenous Hb was present in the plasma:
where DO2 is oxygen delivery, CI is cardiac index, and CaO2 and CvO2 are arterial and venous content, respectively (CaO2 and CvO2 = [HbO2%/100] × total hemoglobin (tHb) × γ [where γ is oxygen capacity for rat or bovine Hb; OSM3 programmed algorithms]).
For all groups, means (±SEM) are reported. Statistical comparisons between groups were initially analyzed with a multifactorial (fraction of inspired oxygen, treatment) with repeated measures (time) ANOVA (). If this analysis revealed differences among groups for baseline values, an analysis of covariance was performed, and adjusted means for 60 and 120 minutes were generated and evaluated. The Tukey-Kramer multiple comparison was used to identify differences between group means. Percent changes from baseline were calculated for key variables, and a single-factor ANOVA with a Tukey-Kramer multiple comparison test was used to determine statistical differences among groups. Statistical analyses were performed using JMP version 5 statistical software package (SAS Institute, Cary, North Carolina) with statistical significance set at a P value of 0.05 or less.
BLOOD GAS RESPONSES IN TREATED AND UNTREATED RATS EXPOSED TO EITHER NORMOXIC OR ACUTE HYPOXIC CONDITIONS
NORMOXIC RATS SYSTEMIC HEMODYNAMIC RESPONSES TO S-NITROSYSOLATED POLYMERIZED BOVINE HEMOGLOBIN
NORMOXIC RATS PULMONARY VASCULAR HEMODYNAMIC RESPONSES TO S-NITROSYSOLATED POLYMERIZED BOVINE HEMOGLOBIN
HYPOXIC RATS SYSTEMIC HEMODYNAMIC RESPONSES TO S-NITROSYSOLATED POLYMERIZED BOVINE HEMOGLOBIN