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Am J Physiol Heart Circ Physiol 284: H81-H91, 2003. First published September 26, 2002; doi:10.1152/ajpheart.00562.2002
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Vol. 284, Issue 1, H81-H91, January 2003

Sodium selenite reduces hemoglobin-induced venular leakage in the rat mesentery

Ann L. Baldwin1, Elizabeth B. Wiley1, Amber G. Summers2, and Abdu I. Alayash2

1 Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona 85724-5051; 2 Laboratory of Plasma Derivatives, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Modified Hbs are being developed as "blood substitutes," but intravascular injection of diaspirin cross-linked Hb (DBBF-Hb) can produce venular leakage. Hb toxicity may arise from reactive oxygen species, so the antioxidant sodium selenite (Na2SeO3) was used in an attempt to reduce leak formation. In anesthetized Sprague-Dawley rats, one-half of which received 2 × 10-6 g/ml Na2SeO3 in their drinking water for 3 wk, the mesenteric microvasculature was perfused with 2 mg/ml DBBF-Hb (N = 8) for 10 min. Controls (N = 7) received saline. This was followed by perfusion with FITC-albumin for 3 min, fixation, and microscopic examination. In rats given DBBF-Hb, Na2SeO3 significantly reduced leak number, leak area, and mast cell degranulation. Venular leakage was also reduced in rats that only received Na2SeO3 locally during DBBF-Hb perfusion. However, Na2SeO3 did not affect animals receiving cyanomet-DBBF-Hb instead of DBBF-Hb and significantly increased leak number and mast cell degranulation in animals receiving saline. In vitro, Na2SeO3 reduced the oxidation rate of DBBF-Hb while in the presence of oxidants. These results suggest that Na2SeO3 reduces DBBF-Hb-induced microvascular leakage partly by retarding the oxidation of its heme iron.

blood substitutes; fluorescence microscopy; mast cells; antioxidant


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HB-BASED OXYGEN CARRIERS, such as diaspirin cross-linked Hb (DBBF-Hb) and polyethylene glycol (PEG)-conjugated Hb, have been proposed as blood substitutes for transfusions due to their plasma expansion and oxygen transport capabilities. Previously, we showed that a bolus injection of PEG-conjugated Hb or DBBF-Hb caused increased microvascular permeability and mast cell (MC) degranulation in a rat mesenteric preparation that required anesthesia, ventilation, a several-minute exposure of tissue to possibly hypoxic conditions, and the excitation of a fluorescent tracer (7). Control preparations that were not injected with Hb-based oxygen-carrying solutions showed significantly lower levels of microvascular permeability and MC degranulation. It is disadvantageous for a potential blood substitute to cause microvascular leakage, because the substitute itself will rapidly leave the circulation and, in addition, alterations in transvascular exchange of plasma proteins will disturb the fluid balance between blood and tissue. Increased microvascular leakage also changes the kinetics of delivery of intravascularly injected drugs, and of endogenous enzymes and hormones, to various tissues. When transfusions are needed, for example, after hemorrhagic shock, it is important that regulation of microvascular exchange is not compromised.

One property of Hbs that may account for their deleterious effects on the microcirculation is the fact that they undergo autoxidation and oxidative redox side reactions (2, 14, 17, 31). Reactive oxygen species (ROS), such as superoxide (O<UP><SUB>2</SUB><SUP>−</SUP></UP>·) and hydrogen peroxide (H2O2), are byproducts of autoxidation. Some Hb-based blood substitutes have been shown to oxidize more readily than Hb in red blood cells in response to chemical modifications aimed at lowering oxygen affinity (40). The reaction between oxy, or ferric, forms of Hb with H2O2 produced by autoxidation, or by neutrophils and macrophages, proceeds via the highly reactive ferryl intermediate HbFe(IV) and may lead to heme degradation and cytotoxicity (1). Another possible redox mechanism involves the release of free iron from Hb after oxidative damage. The free iron then catalyzes the reaction between H2O2 and O<UP><SUB>2</SUB><SUP>−</SUP></UP>· to form the hydroxyl radical (OH·) (21). However, Simoni et al. (34) found that when Hb was reacted with H2O2 in vitro, from 15 min to 24 h, very little Fe was released. Therefore, either modified Hbs are less stable in vivo than they are in vitro or else most ROS are produced via the ferryl radical, HbFe(IV).

It has been shown that supplementation with the antioxidant Na2SeO3 can prevent the structural damage and disruption of cellular integrity caused by peroxidation of membranes and other cell components (36). One possible mechanism for the protective effect of Na2SeO3 is that it is required for activation of glutathione peroxidase (35), which is a tetrameric enzyme present in the cytosol that reduces H2O2 as well as fatty acid hydroperoxides. Thus glutathione peroxidase provides protection against oxidative damage and accumulation of free radical products.

Simoni et al. (33) demonstrated that treatment with Na2SeO3 was very effective in the prevention of oxidative damage induced by Hb. In these experiments, lipid peroxidation was evaluated by measuring the formation of conjugated dienes and thiobarbituric acid reactants. Both products were significantly increased in the liver, heart, and plasma of control rats after injection of modified bovine Hb but not in the plasma and tissues of rats that received a dietary supplement of Na2SeO3. In addition, injection of Hb caused histological changes in the liver and myocardium of control rats but not in rats that received Na2SeO3.

Experiments were designed to determine whether preadministration of the antioxidant Na2SeO3 would reduce the DBBF-Hb-induced microvascular leakage to albumin and MC degranulation observed in our rat mesenteric preparation. For comparison, some experiments were also performed with cyanomet-DBBF-Hb (CNmet-DBBF-Hb), a Hb in which the CN groups are tightly bound to the heme such that the molecule is unable to participate in redox reactions. The mesentery is an excellent preparation for quantifying the degree of vascular leakage invoked by particular mediators because the tissue is very thin and the microvascular networks are almost two dimensional, thus facilitating image acquisition and analysis. We have previously used the mesentery to detect venular leaks to albumin after administration of histamine (8, 37) or nitric oxide synthase inhibitors (9). To aid in the interpretation of the results of the animal experiments, the interactions of Na2SeO3 with DBBF-Hb and CNmet-DBBF-Hb, in which the met-DBBF-Hb is trapped with cyanide, were investigated in vitro. Rates of autoxidation and oxidation of the Hbs were measured in the presence and absence of Na2SeO3. In addition, the effect of Na2SeO3 on the oxygen affinity of DBBF-Hb was determined.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preexperimental Treatment of Rats

Male Sprague-Dawley rats, weighing 300-350 g, were obtained from Harlan Teklad (Madison, WI). Monthly serology, bacteriology, and parasitology evaluations are performed on animals from each virus-free barrier at Harlan Teklad. The rats were transported to the animal facility at the Tucson Veterans Administration Medical Center by truck. The animal facility is small with a low personnel activity, and monthly tests are performed on sentinel rats. On arrival, the animals were housed two per cage in a room (3 × 4 m) deliberately chosen so as to be remote from noisy air vents, cage washers, etc. The cages were 45 cm long × 24 cm wide and contained standard Harlan Sanichip bedding. Ten to twenty rats were housed in the room at any given time, and no other rats, apart from those participating in this study, were housed with them. A technician entered both rooms once a day to feed and tend to the rats. The temperature ranged between 22 and 23°C, and the humidity was kept between 55 and 60%. The rats were fed Harlan Tech Lab 485 rat chow and placed on a light cycle with lights on between 6:00 AM and 6:00 PM.

Sodium Selenite Pretreatment

One-half of the rats received 2 µg/ml Na2SeO3 (Sigma; St. Louis, MO) in their drinking water (2 ppm). Na2SeO3 has previously been administered to rats in their drinking water at a dose of 0.1 µg/ml and has been shown to be effective in increasing antioxidant enzyme activities (42). In the present experiments, each rat drank an average of 32 ml water/day; therefore, each rat received 64 µg Na2SeO3 daily. In addition, each rat received 5-6 µg Na2SeO3 from the diet. Sodium selenite-treated rats were kept as described for 3 wk before experiments were performed. Non-Na2SeO3-treated rats were kept for 3 wk under the same conditions but given tap water.

Selenium Evaluation

Experiments were performed on five control and five Na2SeO3-treated rats to determine the concentrations of Se in the blood and intestinal tissue. Each rat was anesthetized with 1 ml of 3:1 ketamine-acepromazine. The abdominal cavity was opened by a midline incision, and the aorta was exposed. An 18-gauge needle was attached to a heparinized syringe, and the needle was carefully inserted, retrograde, into the aorta so that 3 ml of blood could be collected. The blood was placed in three heparinized centrifuge tubes (1 ml in each) after removal of the needle from the syringe to prevent hemolysis. The tubes were then centrifuged, and the plasma was separated for storage at -80°C. Next, the distal and proximal ends of the small intestine were clamped, as were the mesenteric artery and vein. The entire small intestine was then excised and divided into three segments. The intestinal contents were removed, and each segment was wrapped in tinfoil, labeled, and placed on dry ice. The animals were euthanized with an injection of Beuthanasia in the jugular vein. Plasma and tissue samples were packed in dry ice and sent to Oscar E. Olson Biochemistry Laboratories, South Dakota State University, for analysis of Se content using the fluorometric method (12).

Hemoglobin Solutions

DBBF-Hb. Human Hb cross-linked by bis(3,5-dibromosalicyl)fumarate (DBBF-Hb) was a kind gift from the Walter Reed Army Institute of Research (Washington, DC). A spectral analysis of Hb oxidation products was performed using a Beckman DU640 spectrophotometer. The ratio of oxy (Fe2+) to met (Fe3+) forms of Hb was calculated as previously described (38a) and found to be 0.21. DBBF-Hb was filtered using a Sephadex G-50 column that was equilibrated with HEPES-buffered saline (HBS; pH 7.4). The components of the HBS were as follows (in mM): 10 HEPES sodium salt, 11 HEPES acid, 132 NaCl, 4.7 KCl, 2.0 CaCl2, and 1.2 MgSO4, giving a total molarity of 160.9 mM and a total osmolarity of 323.8 mosM. To increase the relative concentrations of oxyHb to metHb, DBBF-Hb was reduced by adding sodium dithionite (50 mg/100 ml). The sample was then immediately applied to a Sephadex G-50 column, and the eluant, recognized as reduced Hb by its cherry-red color, was collected. Spectral analysis of this solution demonstrated that it contained 1.2% metHb, which is acceptable.

Met- and CNmet-DBBF-Hb. The met (ferric) form of DBBF-Hb was prepared by oxidizing ferrous Hb with an excess amount of potassium ferricyanide. The reaction products were passed through a Sephadex G-25 column in 50 mM phosphate buffer (pH 7.4), 1 M NaCl to remove unreacted ferricyanide. Sephadex G-25 columns equilibrated in 50 mM phosphate buffer (pH 7.4) were subsequently used to remove salt in the metHb solutions (3). Treatment of met Hb with potassium cyanide results in the formation of the ferric cyanide derivative (cyanometHb). Cyanide is a strong ligand with respect to metHb, giving highly stable cyanomet Hb. We used the met form of DBBF-Hb to prepare CNmet-DBBF-Hb as previously described (4). Briefly, 1 vol of 1 mM DBBF-Hb solution was mixed with 2 vol of Drabkin reagent. [This reagent, normally used for determination of Hb in blood, is prepared by dissolving 1 g NaHCO3, 50 mg KCN, and 200 mg K3Fe(CN)6 in water, bringing the volume to 1 liter.] After a 5-min incubation, the mixture was passed through two successive cycles of Sephedex G-25 columns to remove excess ferri and any bound ferrocyanide. Millimolar extinction at 540 nm of 12.5 mM-1 · cm-1 was used to calculate the concentration of CNmet-DBBF-Hb.

Design of Animal Experiments

The following study was performed to characterize the mesenteric microvascular leakage to albumin caused by intravenous injection of DBBF-Hb in control and Na2SeO3-treated rats. Four different protocols were performed: DBBF-Hb perfusion with and without Na2SeO3 pretreatment (6 and 5 rats, respectively) and HBS with 0.5 g/100 ml BSA (HBS-BSA) perfusion with and without Na2SeO3 pretreatment (3 and 5 rats, respectively). In a fifth group (6 rats), which did not received Na2SeO3, the microvasculature was perfused for 10 min with DBBF-Hb plus 2 µg/ml Na2SeO3. The same concentration of Na2SeO3 was also added to the HBS-BSA suffusate that was applied during the 10-min perfusion (Na2SeO3 drip experiments). For comparison, two more groups of experiments were performed with CNmet-DBBF-Hb, a Hb in which the CN groups are tightly bound to the heme such that the molecule is unable to participate in redox reactions. In one group (5 rats), CNmet-DBBF-Hb was perfused in the mesenteric microvasculature as described for DBBF-Hb. In the other group (5 rats), Na2SeO3 was added to the CNmet-DBBF-Hb perfuste and to the suffusate. The number of animals per group was justified to be sufficient by utilizing a sample size nomogram in conjunction with estimates of the difference in means that needed to be detected and the means ± SD for each parameter (40).

Surgical Procedures

The animal procedure was similar to that described previously (8) and is summarized here. Thirty-five male Sprague-Dawley rats (350-400 g) were preanesthetized with 1 mg/kg body wt of the following mixture: ketamine hydrochloride (5 ml of 100 mg/ml), acepromazine maleate (2 ml of 10 mg/50 ml), and xylazine (8 ml of 20 mg/ml). This was followed by an intraperitoneal injection of pentobarbital sodium (30 mg/kg). In each rat, a tracheostomy was performed for artificial ventilation. The abdomen was opened, and several contiguous well-vascularized mesenteric windows were selected and spread out flat over a Plexiglas platform. The superior mesenteric artery was cannulated near the selected mesenteric windows, and the appropriate arterioles and venules bordering the windows were ligated to allow perfusion of only the chosen windows (Fig. 1).


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  		Fig. 1.   Diagram to illustrate the mesenteric window preparation. The mesenteric artery was cannulated, proximal to the feeding arterioles of the chosen window. The circulation of the window was then effectively isolated from the rest of the mesenteric circulation by ligating the arteriole and venule, bordering the intestine, on each side of the window. The microvasculature of the window was perfused free of blood, and the mesenteric artery was clamped distal to the window to maintain microvascular pressure.

The mesenteric windows were flushed clear of blood with HBS-BSA (pH 7.4) and 1 U/ml heparin at 37°C and perfused, at an inlet pressure of 100 mmHg, with this solution alone or with an additional 2 mg/ml DBBF-Hb or CNmet-DBBF-Hb for 10 min. The mesenteries were kept moist by suffusion with HBS-BSA at 37°C. In experiments on 11 rats that had not received Na2SeO3 in their water, the perfusate (DBBF-Hb or CNmet-DBBF-Hb) and the suffusate contained 2 µg/ml Na2SeO3, and 20 ml of the suffusate was used during the 10-min perfusion. The perfusate was then replaced by FITC-albumin (Sigma) in HBS-BSA for the next 3 min. As soon as the vasculature of the windows was filled with FITC-albumin, as judged by the color, the pressure was adjusted to 40 mmHg, and the portal vein, which acted as the flow outlet, was clamped. After 3 min, the clamp was removed, and 3 ml fixative (3% formaldehyde in HBS) was perfused via the cannula at a pressure of 100 mmHg. Next, the pressure was reduced to 40 mmHg, the portal vein was clamped, and fixation was continued for 60 min at 4°C. The mesenteric tissue was carefully excised, and two windows from each preparation were mounted between two thin glass coverslips using aqueous mounting medium (Vectashield, Vector Laboratories; Burlingame, CA). The remaining mesenteric windows from each experiment were spread flat on microscope slides and suffused with 1 g/100 ml toluidine blue for 20 s before being mounted. Toluidine blue was used to stain the MCs to determine the numbers that had degranulated. Degranulated mesenteric MC (DMC), identified by the presence of intracellular granules released into the surrounding tissue, were counted within each circular ×20 field of view of a Zeiss Axioplan light microscope (field area 1.13 mm2). Rows of fields were counted systematically from left to right. Cells located in the periphery of the field were only counted if at least one-half of the cell area was within the field. The error of repeat counting was <2%. About 30 fields were counted for each mesenteric window.

Assessment of Venular Leakage

An assessment of overall vascular leakage was made by measuring the number and area of regions with extravascular FITC-albumin. Slides were examined microscopically using a ×10 objective (numerical aperture 0.6). The light source was a 100-W Hg lamp for epifluorescence and a halogen lamp for transmitted illumination. A videocamera (Optronix 750D) was mounted at the camera port of the microscope. Five images of leaky vessel networks from each mesentery, produced by epifluorescence with the appropriate FITC excitation and emission filters (wavelength = 488 and 515 nm, respectively), were viewed on a black-and-white monitor and also recorded on a videorecorder. Each slide was only exposed to the excitation wavelength for 5 s. Recordings were also made of the networks under transillumination. Videotaped images were later analyzed using an analog-to-digital converter and appropriate software (NIH Image) to measure the length and diameter of each venule, number of leaks per venule, and area of each leak. If a leak was positioned at a vascular junction, the leak area was divided by the number of venules involved. Data were pooled within each group (i.e., non-Na2SeO3, DBBF-Hb), and the following values were calculated: 1) average number of leaks per length of venule, and 2) average leak area per micrometer of venule.

Statistical Analysis for Animal Experiments

Each parameter was compared between different groups using one-way ANOVA. If a significant difference was found between groups, pairs of groups were compared using the Student t-test with a P value <0.01 to determine statistical significance. All values are presented as means ± SE. The N used in the selenium evaluation studies was the number of rats because the plasma selenium concentration is a systemic measurement. The n used in the leakage studies was the number of venules examined per group, and that used for the mast cell degranulation was the number of mesenteric fields of view examined per group, both of these quantities being the units of variation.

Effect of Na2SeO3 on the Oxidation of DBBF-Hb and CNmet-DBBF-Hb

Autoxidation experiments. Autoxidation experiments for DBBF-Hb samples (50 µM) in HBSS (pH 7.4, 37°C) in the presence of 500 µM Na2SeO3 were carried out in the dark. Absorbance changes in the range of 490-640 nm due to the spontaneous oxidation of DBBF-Hb were recorded on a Perkin Elmer Lamda 6 spectrophotometer. Multicomponent analysis was used to calculate the time-dependent changes in concentration of the oxy, met, and deoxy forms based on the known spectra of each species (26). First-order autoxidation rate constants were derived from a fit of the data during 6-h incubation to a single exponential expression using a nonlinear least-squares fitting program (Origin 6.1).

Enzymatic oxidation experiments. Oxidation of DBBF-Hb by low and steady levels of H2O2 was achieved in vitro with the use of the glucose/glucose oxidase system (GOX) as previously described (20). The reaction mixture was prepared containing 50 µM DBBF-HbFe2+ or DBBF-HbFe3+CN and 10 mU/ml glucose oxidase in HBSS solution (pH 7.4), which also contained 5.6 mM glucose as substrate at 37°C. Under these conditions, the rate of H2O2 production by the GOX system was 1.5 µM H2O2/min (20). In another set of experiments, 500 µM Na2SeO3 was added to the reaction mixture and spectral changes in the Hb were monitored throughout the experiments.

Selenium-mediated reduction experiments. In these experiments, either the met or cyanomet derivatives of DBBF-Hb (50 µM) were incubated in HBSS buffer (pH 7.4, 37°C) with increasing concentrations of selenium (125 µM-1 mM). The reduction of the ferric iron of DBBF-Hb induced by selenium was monitored immediately after mixing of the Hb solutions and selenium in a cuvette placed inside a diode array spectrophotometer.

Effect of Na2SeO3 on the Oxygen Affinity of DBBF-Hb

Oxygen equilibrium curves of DBBF-Hb in the presence of various concentrations of Na2SeO3 (250 µM-1 mM) were obtained using the Hemox Analyser (TCS Scientific; New Hope, PA). This instrument measures the oxygen tension with a Clark Oxygen Electrode (model 5331 Oxygen Probe, Yellow Springs Instrument; Yellow Springs, OH) and simultaneously uses a dual-wavelength spectrophotometer to calculate the Hb oxygenation. Oxygen equilibrium experiments were carried out in 0.1 M phosphate buffer (pH 7.4), and the incubation times ranged from 10 to 20 min. The concentration of Hb samples was between 60 and 75 µM (heme), and the temperature was maintained at 37°C. To maintain the metHb content to a minimum level (<2%), 4 µl of the Hayashi enzymatic reduction system, which consists of a number of red blood cell enzymes and cofactors, were included in the final solution (4 ml) (23). In another set of experiments, the Hayashi enzymatic system was removed to determine the effects of increasing concentrations of selenium on met Hb build up throughout the experiments and consequently the oxygen affinity of DBBF-Hb. Oxygen equilibrium parameters were derived by fitting the Adair equations to each oxygen equilibrium binding curve by the nonlinear least-squares procedure included in the Hemox Analyzer software (p50 PLUS, version 1.2) (3).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Selenium Evaluation

The amount of Se in plasma of rats that were not given Na2SeO3 in their drinking water was 0.496 ± 0.011 (mean ± SE) µg/ml (N = 4). Rats given exogenous Na2SeO3 showed significantly more Se in their plasma [0.571 ± 0.010 (mean ± SE) µg/ml (N = 4)]. The corresponding values for intestinal tissue were 0.363 ± 0.027 (mean ± SE) (N = 4) and 0.460 ± 0.038 (mean ± SE) µg/ml (N = 4); the Na2SeO3-treated rats showed significantly more Se in their tissue. The basal value for Se in plasma is close to that obtained from previous studies in which rats were fed a diet with a standard content of Na2SeO3 (26, 27). These values were 0.414 ± 0.038 (mean) and between 0.480 and 0.516 µg/ml (SD), respectively. Administration of exogenous Na2SeO3 increased the plasma concentration of Se by 15% in this study, which is identical to the result from a previous study (34) in which rats received 5 µg/kg body wt Na2SeO3 in their drinking water for 7 days, followed by an intramuscular injection of 5 µg/kg body wt Na2SeO3. Administration of exogenous Na2SeO3 increased the concentration of Se in the intestine by 27% in this study. No values for Se concentrations in intestinal tissue could be found in the literature.

Distribution of Leaks

Microscopic examination of control mesenteric preparations (non-Na2SeO3, HBS-BSA) by epifluorescence revealed very few leaky sites. However, non-Na2SeO3 preparations treated with DBBF-Hb showed many leaks (Fig. 2). The leakage occurred in venules but not in arterioles or capillaries. The total numbers of venules examined for non-Na2SeO3, HBS-BSA and non-Na2SeO3, DBBF-Hb preparations were 207 and 299, respectively, and the percentages of venules demonstrating leaks for these two categories were 2.5% and 38.4%, respectively. Most of the leaks were <100 µm2 in area; 93.3% in the case of non-Na2SeO3, HBS-BSA and 94.2% in the case of non-Na2SeO3, DBBF-Hb preparations. This compares with a luminal surface area of endothelial cells that ranges from 400 to 900 µm2 (8). Rats given drinking water containing Na2SeO3 showed decreased DBBF-Hb-induced mesenteric microvascular leakage to FITC-BSA compared with rats that did not receive Na2SeO3. A typical network from a Na2SeO3 rat after perfusion with DBBF-Hb, followed by FITC-BSA, can be seen in Fig. 3. No leakage sites of FITC-BSA in the microvasculature are visible. Inclusion of Na2SeO3 in the DBBF-Hb perfusate and the HBS-BSA suffusate during DBBF-Hb perfusion (Na2SeO3 drip) had a similar but less pronounced effect on DBBF-Hb-induced venular leakage compared with that caused by long-term Na2SeO3 pretreatment. The total numbers of venules examined for Na2SeO3 pretreatment DBBF-Hb and Na2SeO3 drip DBBF-Hb preparations were 270 and 337, respectively, and the percentage of venules demonstrating leaks for these two categories were 2.6% and 3.1%, respectively. Most of the leaks were <100 µm2 in area (100% for Na2SeO3, DBBF-Hb and 92.5% for Na2SeO3 drip, DBBF-Hb preparations).


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  		Fig. 2.   Light microscopic image of mesenteric microvasculature from a non-Na2SeO3 rat after perfusion with diaspirin cross-linked Hb (DBBF-Hb), followed by FITC-albumin. Note the leakage sites of FITC-albumin from the venules. Scale bar = 100 µm.



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  		Fig. 3.   Light microscopic image of mesenteric microvasculature from Na2SeO3 rat after perfusion with DBBF-Hb followed by FITC-albumin. No leakage sites of FITC-albumin from the microvasculature are visible. Scale bar = 100 µm.

Dietary selenium had the opposite effect on mesenteries that were perfused with HBS-BSA rather than with DBBF-Hb before perfusion with FITC-BSA; venular leakage was more than doubled by pretreatment with Na2SeO3. The total number of venules examined in the Na2SeO3, HBS-BSA case was 313, and 14.7% of those venules contained leaks compared with 2.5% in the non-Na2SeO3, HBS-BSA case.

In rats whose mesenteric microcirculation was perfused with CNmet-DBBF-Hb, only 5.2% of the 303 venules examined showed leaks. Inclusion of Na2SeO3 in the perfusate and suffusate during perfusion with CNmet-DBBF-Hb reduced the percentage of leaky venules to 1% of 295 venules.

Quantification of Leaks

The mean numbers and areas, respectively, of leaks per venule length for networks from Na2SeO3 and non-Na2SeO3 rats perfused with HBS-BSA, DBBF-Hb, or CNmet-DBBF-Hb are shown in Fig. 4, A and B. In rats given DBBF-Hb, Na2SeO3 significantly reduced the microvascular leak number from 2.41 ± 0.33 × 10-3 to 0.08 ± 0.03 (means ± SE) × 10-3 leaks/µm venule length and the area from 0.10 ± 0.03 to 0.0008 ± 0.0004 µm2/µm (P < 0.01). A value of 0.08 × 10-3 leaks/µm means that one leak was found in 12,500 µm of venule length. A typical total sum of venule lengths measured in one preparation was ~30,000 µm. Venular leakage, induced by DBBF-Hb, was also significantly reduced in rats that did not receive Na2SeO3 in their drinking water but had their mesenteries perfused and suffused with Na2SeO3. In this case, the leak number was reduced to 0.23 ± 0.09 × 10-3 leaks/µm venule length (P < 0.01) and the area to 0.012 ± 0.006 µm2/µm (P < 0.01). In rats perfused with HBS-BSA, pretreatment with Na2SeO3 in the drinking water increased the number of leaks per micrometer of venule length from 0.12 ± 0.08 × 10-3 to 0.78 ± 0.14 × 10-3 (P < 0.01). The mean leak area was increased, but not significantly, from 0.02 ± 0.02 to 0.06 ± 0.02 µm2/µm. In rats perfused with CNmet-DBBF-Hb, without the addition of Na2SeO3, the mean leak number per micrometer (0.36 ± 0.14 × 10-3 leaks/µm venule length) and the mean leak area per micrometer (0.005 ± 0.003 µm2/µm) were significantly lower than after DBBF-Hb perfusion but were not significantly different from controls. Inclusion of Na2SeO3 in the perfusate and suffusate during the 10-min perfusion did not significantly affect the results. The corresponding values were 0.53 ± 0.32 × 10-3 leaks/µm venule length and 0.007 ± 0.004 µm2/µm.


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  		Fig. 4.   A: average number of leaks per venule length (in leaks/µm venule length) for different treatments. B: average total leak area per venule length (in µm2/µm) for different treatments. Error bars indicate SE. Significant differences are as follows: * greater than the corresponding control group; and ** smaller than the corresponding control group. S, HEPES-buffered saline (HBS)-BSA; S/SeWater, HBS-BSA with Na2SeO3; Hb, DBBF-Hb; Hb/SeWater, DBBF-Hb with Na2SeO3 pretreatment; Hb/Se, DBBF-Hb with Na2SeO3; CHb, cyanomet (CNmet)-DBBF-Hb; CHb/Se, CNmet-DBBF-Hb with Na2SeO3.

MC Degranulation

DMC were easy to identify in mesenteric tissue stained with Toluidine blue because the granule contents were expelled into the surrounding tissue and were stained a dark red against a blue background. Intact MC formed compact, oval-shaped disks that also stained dark red. Intact MC and DMC are shown in Fig. 5, A and B, respectively. The intact MC are from a non- Na2SeO3, HBS-BSA-perfused preparation, and the DMC are from a Na2SeO3, HBS-BSA-perfused preparation. The number of mesenteric windows per group examined for MC degranulation was 6 except for the Na2SeO3, HBS-BSA group, in which 11 windows were examined because this group showed a great variation in the measurements obtained. The numbers of fields of view observed were 83, 161, 74, 93, and 106 for HBS-BSA; Na2SeO3, HBS-BSA; DBBF- Hb; Na2SeO3, DBBF-Hb; and Na2SeO3 drip, DBBF-Hb preparations, respectively. In the HBS-BSA-perfused animals, Na2SeO3 significantly increased the number of DMC per field of view from 8.24 ± 1.09 to 25.43 ± 1.86 (means ± SE, P < 0.01; Fig. 6). Non-Na2SeO3 rat mesenteries that were perfused with DBBF-Hb showed ~50% more MC degranulation than those perfused with HBS-BSA (12.88 ± 1.61 vs. 8.24 ± 1.09), and this difference was statistically significant (P < 0.01). In preparations that were perfused with DBBF-Hb, long-term pretreatment with Na2SeO3 significantly decreased the number of DMC per field of view from 12.88 ± 1.61 to 1.99 ± 0.52 (means ± SE, P < 0.01). However, when Na2SeO3 was just added to the perfusate and suffusate during administration of DBBF-Hb, the MC degranulation was not reduced (13.86 ± 1.20, P < 0.01). Mesenteric MC degranulation in animals whose mesenteric microvasculature was perfused with CNmet-DBBF-Hb (6.85 ± 0.78 per field of view) was significantly lower than that for the non-Na2SeO3, DBBF-Hb group and control group. Inclusion of Na2SeO3 in the perfusate and suffusate did not affect the results (6.06 ± 0.72 per field of view).


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  		Fig. 5.   A: light micrograph showing mesentery stained with Toluidine blue to reveal mast cells (MCs). In this field of view, from a non-Na2SeO3, HBS-BSA-perfused preparation, the MCs are intact. B: in this field of view, from a Na2SeO3, HBS-BSA-perfused preparation, the MCs are degranulated (DMC).



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  		Fig. 6.   Average number of DMC per microscopic field of view for different treatments. Significant differences are as follows: * greater than the corresponding control group; and ** smaller than the corresponding control group. Abbreviations are as for Fig. 4.

Effect of Na2SeO3 on the Oxidation of DBBF-Hb and CNmet-DBBF-Hb

We first monitored the effects of inclusion of 500 µM Na2SeO3 with DBBF-Hb as it undergoes normal spontaneous oxidation under physiological conditions in HBSS buffer (pH 7.4, 37°C) for 6 h. The calculated rates of autoxidation of DBBF-Hb with and without Na2SeO3 were unchanged and were close to an earlier reported value of 0.0240 h-1 (30). However, there was an ~20-30% reduction in the rates of autoxidation of DBBF-Hb when red blood cell antioxidant enzymes, such as superoxide dismutase (O<UP><SUB>2</SUB><SUP>−</SUP></UP>· scavenger) and catalase (H2O2 scavenger) were included in the buffer medium (data not shown), which led us to believe that Na2SeO3 under these experimental conditions lacks a strong antioxidant effect. We then focused on the effects of Na2SeO3 on DBBF-Hb oxidation under mild oxidative stress induced by low and steady supplies of H2O2, at a rate of 1.5 µM/min generated by the glucose/glucose oxidase system (20). Figure 7 shows typical time courses of the oxy disappearance and met-DBBF-Hb buildup as a function of time in the presence of the GOX system and in the presence or absence of Na2SeO3. The rate for the exponential decrease in oxy-DBBF-Hb was calculated to be 0.0298 min-1 (Fig. 7A). However, in the presence of Na2SeO3, the rate of oxy Hb decay was 0.0155 min-1, ~50% slower than Hb/GOX medium alone (Fig. 7B). Similarly, the rate of met Hb formation in the presence of Na2SeO3 was 0.0126 min-1, ~70% lower than 0.0350 min-1 calculated for Hb/GOX alone. To investigate the potential direct redox interaction between Hb oxidation forms and selenium, we monitored the effect of selenium incubation with the met and cyanomet forms of DBBF. The dependence of metHb reduction by selenium at various concentrations is shown in Fig. 8. It is clear that as the concentration of selenium increases, there was a corresponding increase in the reduction in the levels of metHb in solutions. The rapid reduction in met-DDBF-Hb, even at a low selenium concentration, is not surprising because selenium occupies a higher position in the pecking order of free radicals than iron in terms of standard reduction potential and is therefore capable of donating electrons to the ferric iron of Hb (12). However, similar concentrations of selenium showed no effects on the levels of metHb trapped by cyanide, confirming the need for an unliganded binding site for the redox communication to occur between selenium and the ferric iron of DBBF-Hb.


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  		Fig. 7.   A: time-dependent changes in the redox states of DBBF-Hb (50 µM) [oxy () and met (open circle ) forms] in the presence of the glucose/glucose oxidase (GOX) enzymatic system (10 mU/ml). Other oxidation products such as ferryl/hemichromes are not shown. Visible absorbance spectra were collected every 5 min for 2 h, and the changes in the oxidation states of Hb were monitored. B: time-dependent changes in the redox states of DBBF-Hb in the presence GOX (conditions as in A) plus selenium (500 µM). The plots in A and B represent the experimental data, whereas the solid lines correspond to the best exponential fits using SigmaPlot.



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  		Fig. 8.   Percent reduction in met-DBBF-Hb (%MetHb) in the presence of Na2SeO3. Met-DBBF-Hb (50 µM) was incubated with increasing concentrations of selenium, which varied from 125 µM to 1 mM in HBSS buffer at room temperature. Each bar represents the mean ± SE of three independent experiments.

Effect of Na2SeO3 on the Oxygen Affinity of DBBF-Hb

Increasing concentrations of sodium selenite did not appear to affect the oxygen affinity of DBBF-Hb, as demonstrated by the constancy of values of the PO2 at which Hb in 50% saturated (P50) and the cooperativity (Hill number) in the presence or absence of reducing enzymes (Table 1). In the presence of enzymes, an average P50 value of 29.4 mmHg was obtained for DBBF-Hb regardless of the selenium concentrations used. This value is close to that calculated for Hb solutions without selenium (control; P50 = 29.0 ± 1.1 mmHg). In the presence of selenium (125 µm-1 mM) alone, however, the average P50 was calculated to be ~23.05 ± 0.09 mmHg. The decline in the P50 value was most likely due to the accumulation of met Hb throughout the experiments (~8-9% of total heme).

                              
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  		Table 1.   Effect of Na2SeO3 on oxygen affinity of DBBF-Hb


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study has demonstrated that chronic administration of Na2SeO3 in the drinking water of rats can significantly reduce microvascular leakage and MC degranulation induced by DBBF-Hb in our mesenteric preparation and that short-term, localized application of Na2SeO3 to the blood vessels and tissue is almost as effective. It should be noted that the concentration of locally applied Na2SeO3 was significantly higher than that in the plasma of Na2SeO3-pretreated animals (2 mg/ml vs. 0.57 µg/ml). In addition, the locally applied Na2SeO3 was mainly in the form of selenite, whereas the selenium in the plasma and tissues of chronically treated animals is likely to be present in different forms as well (i.e., plasma selenoproteins). When HBS-BSA was substituted for the DBBF-Hb, Na2SeO3 had the opposite effect; venular leakage and MC degranulation, both of which were normally low in control rats after perfusion with HBS-BSA, increased if the rats were given Na2SeO3.

The reduction in DBBF-Hb-induced venular leaks and MC degranulation, caused by pretreating the animals with Na2SeO3, is consistent with Na2SeO3 acting as an antioxidant and reducing the deleterious effects of ROS. In addition, Na2SeO3 can become incorporated into plasma proteins to form selenoproteins, such as selenoprotein P (SeP). This molecule has been shown to associate with endothelial membranes in vivo (15) and might protect the endothelium from oxidative damage. However, in vivo studies demonstrating the role of SeP as a part of the body's defense against oxidative stress are still lacking. Another possibility to account for the protective effect of Na2SeO3 is that it decreases the rate of oxidation of DBBF-Hb, thus reducing oxidative side reactions of Hb and preventing subsequent tissue damage. This latter mechanism is strongly supported by the results obtained in vitro. These in vitro studies clearly show that Na2SeO3, although it did not influence the rate of autoxidation of Hb, nevertheless strongly suppressed the oxidation of Hb brought about by the GOX. This enzymatic system generates steady fluxes of oxidants, namely, H2O2, which may therefore better mimic the nature and levels of oxidative challenge created in the mesentery under our in vivo experimental conditions (10). The fact that the levels of met rather than the cyanomet forms of DBBF were directly reduced by increasing concentrations of Na2SeO once again supports a redox function for Na2SeO under these experimental conditions.

When Na2SeO3 was given as a pretreatment and the rat mesenteric microcirculation was perfused with HBS-BSA, Na2SeO3 did not behave as an antioxidant and did not protect the endothelium, as demonstrated by the fact that it increased venular leakage and caused MC degranulation. It is known that selenium can activate neutrophils and macrophages to produce oxidants (5, 6). Kiremidjian-Schumacher and Stotzky (25) postulated that the amount of selenite that interacts with the surface of immunocompetent cells may alter the surface properties of the cells and affect not only the uptake, retention and efflux of molecules by the cells but also their ability to respond to stimuli. A previous study (24), using mice, showed that exposure to Na2SeO3 in the drinking water stimulated splenic macrophages to produce cytokines and tumor necrosis factor-alpha (24). Tumor necrosis factor-alpha in the blood can stimulate neutrophil adhesion to rat mesenteric venules (28). If similar reactions occur in rats, then reactions of the Se with neutrophils during the pretreatment period could promote their adhesion to mesenteric venules. In our preparations, adherent neutrophils often remain attached to the endothelium after perfusion with HBS-BSA (38). Such neutrophils would be able to react with the Se in the HBS-BSA perfusate to produce ROS. These neutrophils would also provide a source of reduced glutathione (18). Na2SeO3 is metabolized by its interaction with reduced glutathione, and ROS are byproducts of this reaction (32, 39). Reactions such as these could explain the effect of Na2SeO3 on preparations perfused with HBS-BSA, assuming that these reactions are extensive enough to overwhelm the antioxidant properties of Na2SeO3.

In cases in which DBBF-Hb is present, Na2SeO3 interacts with the DBBF-Hb, as shown by our in vitro results, and thus may not be free to stimulate adherent neutrophils. Thus the interaction is synergistic and the prooxidant properties of both molecules are reduced. Other examples of interactions between Hbs and selenium are cited in the literature. For example, whole blood contains selenium bound to Hb (11, 16, 22, 29). Another study (19) demonstrated that concentrations of oxyHb in blood were significantly increased, whereas the concentrations of metHb were decreased, after the administration of selenium dioxide to rats (19). The authors hypothesized that the selenium increased the activity of glutathione peroxidase and that this enzyme was responsible for the reduction of met Hb. However, the results of this study could also be explained by a direct interaction of Na2SeO3 with Hb.

The relatively small degree of microvascular leakage and MC degranulation accompanying perfusion with CNmet-DBBF-Hb versus DBBF-Hb is consistent with the hypothesis that the tissue damage induced by Hbs is caused by their oxidation and accompanying formation of ROS. As mentioned previously, CNmet-DBBF-Hb is a Hb in which the CN groups are tightly bound to the heme such that the molecule is unable to participate in redox reactions. The fact that Na2SeO3 did not significantly affect the microvascular leakage and MC degranulation that accompanied perfusion with CNmet-DBBF-Hb, whereas it increased these responses after HBS-BSA perfusion, suggests that Na2SeO3 interacted with CNmet-DBBF-Hb and thus was not free to stimulate immune cells.

As mentioned previously, intravascular injection of HBS-BSA in Na2SeO3 rats produced MC degranulation. It is unlikely that administration of Na2SeO3 causes MC degranulation in the absence of perfusion with HBS-BSA, because the mesenteries of Na2SeO3 rats that were perfused with DBBF-Hb showed very little MC degranulation. In this case, if Na2SeO3 in the drinking water had caused the MCs to degranulate, they would have had to repair themselves during the 10-min DBBF-Hb perfusion, which is not possible. Thus perfusion with HBS-BSA, together with administration of Na2SeO3, are both required to cause MC degranulation and microvascular leakage. Perfusion with HBS-BSA in Na2SeO3-treated rats may induce leak formation because when blood is diluted with such solutions, the concentration of superoxide anions in the tissue increase. Unlike plasma, these solutions do not contain ROS scavengers, such as catalase and superoxide dismutase (27), and so the scavengers become diluted and excess ROS accumulate, producing venular leakage and MC degranulation. These responses did not occur in rats that had not imbibed Na2SeO3, possibly because the immune cells were not activated and thus were not producing excess ROS.

With regard to the combined administration of Na2SeO3 and modified Hbs to provide a blood substitute that maintains a low level of macromolecular microvascular permeability, the results with the short-term Na2SeO3 application (Na2SeO3 in DBBF-Hb perfusate with Na2SeO3 suffusion) are most relevant. Obviously, if Na2SeO3 had to be administered for 3 wk before the transfusion with the blood substitute, this protocol would not be useful in emergency situations. In this study, short-term administration of Na2SeO3 was almost as effective as long-term use in reducing the numbers of venular leaks and mean leak area, and this result is promising with regard to the clinical use of Na2SeO3 in combination with cross-linked Hb as a blood substitute. In the current experiments involving short-term administration of Na2SeO3 with DBBF-Hb, the mesenteric tissue was also suffused with Na2SeO3. However, suffusion would not usually be an option in a clinical situation, and thus it would be necessary to depend on the Na2SeO3 contained within the transfusion fluid. Even with this limitation, it is likely that the Na2SeO3 would still reduce the microvascular leaks that may be induced by DBBF-Hb because the intravascularly injected Na2SeO3 would immediately react with the DBBF-Hb and would also immediately contact the endothelium and thus be available for protection. Further research is required, however, to determine the rate of disappearance of Na2SeO3 from the plasma, because it may diffuse into the tissue fluids or be taken up by blood cells.

Therefore, to summarize, it appears as though coadministration of Na2SeO3 with DBBF-Hb significantly reduces the problems of DBBF-Hb-induced venular leak formation and MC degranulation in our preparation of the rat mesentery without impairing the oxygen carrying capability of DBBF-Hb. This effect may be due to the fact that Se is required to activate glutathione peroxidase, an enzyme that protects against oxidative damage. However, the results of our in vitro experiments suggest that Na2SeO3 may reduce the rate of degradation of the DBBF-Hb to the met (Fe3+) form, thus decreasing the rate of formation of ROS. The oxidation of Hb(Fe2+) to the nonfunctional met Hb(Fe3+) form is an important concern in the use of Hb as an oxygen-carrying agent. Uncontrolled and spontaneous oxidation of ferrous iron compromises the function, and in some cases the safety, of the infused Hb-based oxygen carriers. The fact that Na2SeO3 increased microvascular leak formation during perfusion with HBS-BSA demonstrates that the balance between the production and removal of ROS may be a critical factor governing the toxicity of Na2SeO3 supplementation. Thus, when using Na2SeO3 clinically, care must be taken to ensure that production of ROS does not overwhelm the biological system.


    ACKNOWLEDGEMENTS

We thank Meera Jain for expertise in collection and analysis of the data and Francine Wood (Food and Drug Administration) for performing some of the oxygen binding studies.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-53047.

Address for reprint requests and other correspondence: A. L. Baldwin, Dept. of Physiology, College of Medicine, Univ. of Arizona, Tucson, AZ 85724-5051 (E-mail: abaldwin{at}u.arizona.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpheart.00562.2002

Received 8 July 2002; accepted in final form 13 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 284(1):H81-H91


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