(Stroke. 1995;26:1431-1437.)
© 1995 American Heart Association, Inc.
Articles |
Circulation of Red Blood Cells Having High Levels of 2,3-Bisphosphoglycerate Protects Rat Brain From Ischemic Metabolic Changes During Hemodilution
From the Department of Neurosurgery, Fukuoka University School of Medicine, and Department of Clinical Chemistry and Laboratory Medicine (N.H.), Kyushu University Faculty of Medicine, Fukuoka, Japan.
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Abstract |
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Background and Purpose We designed the present study to examine the effects of red blood cell oxygen-delivering capacity on ischemic brain metabolism during hemodilution with respect to red blood cell 2,3-bisphosphoglycerate content.
Methods A modification of red blood cell 2,3-bisphosphoglycerate content was achieved by an exchange transfusion of blood in which red blood cells were treated with either phospho(enol)pyruvate or inorganic phosphate in spontaneously hypertensive rats. Hematocrit values of circulating blood were varied from 30% to 20% during transfusion. Brain ischemia was produced in rats by bilateral carotid artery occlusion lasting 60 minutes. The concentrations of ATP and 2,3-bisphosphoglycerate in the blood and the ATP, phosphocreatine, and lactate concentrations in the brain were estimated by an enzymatic method.
Results Red blood cell 2,3-bisphosphoglycerate concentration increased to 200% of the pretransfusion level after the transfusion in which red blood cells were treated with phospho(enol)pyruvate, whereas the concentration decreased to 80% after the transfusion in which red blood cells were treated with phosphate. Red blood cell ATP content did not differ significantly between the phospho(enol)pyruvate- and phosphate-treated groups after transfusion. When hematocrit was approximately 30%, the ischemic brain ATP and lactate contents did not differ between the nonischemic and ischemic groups. However, as hematocrit was reduced to less than 25% the ischemic brain ATP content remarkably decreased and the lactate content substantially increased in the 2,3-bisphosphoglycerate–subnormal red blood cell group. In contrast, the ischemic brain ATP and phosphocreatine contents in the 2,3-bisphosphoglycerate–enriched red blood cell group were preserved and as high as those in the nonischemic group under the same conditions.
Conclusions Cerebral ischemia was compensated with the increment of cerebral blood flow as a result of the reduction of hematocrit to optimal levels, but the extreme hemodilution induced insufficient oxygen supply to the brain tissue, resulting in a more marked impairment of brain metabolism despite an increase in cerebral blood flow. However, even in extreme hemodilution conditions the 2,3-bisphosphoglycerate–enriched red blood cells in circulating blood protected the brain from ischemic metabolic changes. These results suggest that the 2,3-bisphosphoglycerate–enriched red blood cells in the circulating blood may thus compensate for the insufficient oxygen supply in extremely anemic conditions by providing a sufficient supply of oxygen in the face of ischemic insult.
Key Words: 2,3 biphosphoglycerate • rats • cerebral metabolism • hemodilution • oxygen
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Red blood cells deliver oxygen particularly to metabolically active tissues that produce carbon dioxide. Carbon dioxide diffused into RBCs is ionized to H+ and HCO3- by carbonic anhydrase, and the formed HCO3- is exchanged with extracellular Cl- across the cell membrane by the major integral band 3 protein.1 2 3 The exchange of HCO3- with Cl- accelerates intracellular acidification and facilitates oxygen dissociation from hemoglobin. Thus, the active tissue producing more carbon dioxide is able to accept more oxygen from RBCs. The oxygen dissociation capacity in RBCs is equipped with synergistic effects of hemoglobin, metabolism in the cells, and cell membrane functions. RBC function could be manipulated by modification of these essential elements. 2,3-DPG, a glycolytic intermediate, is one of the most effective factors for modulating the oxygen-delivering capacity of RBCs. One molecule of 2,3-DPG binds preferentially to a tetramer of deoxyhemoglobin4 5 6 7 and stabilizes the hemoglobin conformation to the deoxy form. The oxygen affinity of hemoglobin is reduced by binding of 2,3-DPG to hemoglobin.6 The oxygen dissociation curve of RBCs containing high 2,3-DPG shifts to the right. As a result, the oxygen-delivering capacity increases as the 2,3-DPG concentration in RBCs increases under normoxic conditions.6 The 2,3-DPG concentration in RBCs and the P50 both increase in subjects moving from sea level to high altitude (4530 m).8 Many experimental studies have been performed to evaluate the pathophysiological effects of RBCs with a lowered affinity for oxygen, confirming the advantage of a higher P50 value during normoxic or moderately hypoxic conditions but not in severely hypoxic conditions.9 10 11
Hematocrit is a major determinant of blood viscosity. In humans and animals cerebral blood flow increases when hematocrit decreases.12 13 14 15 Cerebral ischemic damage after carotid ligation depends on the balance between the insufficient oxygen supply caused by a low hematocrit and the increased blood flow caused by low blood viscosity.12 13 14 In the present study we examine the effects of RBCs containing a high concentration of 2,3-DPG on rat brain ischemia during hemodilution.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Manipulation of RBC 2,3-DPG and ATP Concentrations by PEP
Experiments were performed on Wistar rats weighing 500 to 600 g. Whole blood was collected into heparin tubes by an abdominal aortic puncture after anesthesia with sodium pentobarbital (50 mg/kg body wt IP). The packed RBCs were separated from the plasma and buffy coat by centrifugation at 3000g for 5 minutes at 4°C. To increase the 2,3-DPG content in the RBCs, these cells were incubated with a PEP solution (mmol: PEP 50, mannitol 28.8, glucose 50, NaCl 20, and adenine 1; pH 6.0) for 60 minutes at 37°C according to the procedures developed by Hamasaki et al.16 17 The 2,3-DPG content in RBCs was decreased to 50% of the preincubation level after incubation in a similar manner with a Pi solution (mmol/L: sodium phosphate 50, mannitol 28.8, glucose 50, NaCl 20, and adenine 1; pH 6.0). RBCs incubated with either PEP or Pi solution were washed twice in phosphate-buffered saline (pH 7.4) and then recombined with their plasma and human plasma protein fraction (Beyel Pharmaceutical Co Ltd). At the same time, hematocrit was also reduced by regulation of the ratio of RBCs to plasma volume. Blood samples were taken for analysis of ATP, 2,3-DPG, and P50 before and after incubation.
Exchange Transfusion of RBCs Containing High or Low 2,3-DPG
Content
Female spontaneously hypertensive rats 4 to 9 months old and
weighing 200 to 250 g were used in these experiments. The rats were
divided into two groups as follows: a 2,3-DPG–enriched RBC group,
which were transfused with blood treated with a PEP solution, and a
2,3-DPG–subnormal RBC group, whose blood was treated with a Pi
solution. All rats were anesthetized with sodium pentobarbital
(40 mg/kg body wt IP) and then mechanically ventilated with 30%
O2 and 70% N2O after undergoing a tracheotomy.
Pancuronium bromide (0.08 mg/kg body wt IV) was given to induce muscle
paralysis. Both femoral arteries were cannulated, one for
recording of mean arterial blood pressure with an
electromanometer and for anaerobic blood sampling to
monitor blood gases and hematocrit and the other for blood withdrawal.
One femoral vein was cannulated for blood infusion. The rat's head was
fixed in a head holder, and rectal temperature was maintained at
approximately 37°C with the aid of a heating lamp. The exchange
transfusion was performed by a double-syringe pump (941, Harvard
Apparatus) set at infusion and withdrawal rates of 0.8
mL/min. The recombined blood was restored at 37°C for 10 minutes
before transfusion and then was infused through the femoral venous
catheter and withdrawn through the arterial catheter. While
mean arterial blood pressure was monitored, a total of 15
mL blood was exchanged. At the same time, hematocrit values were also
varied from 30% to 20% by exchanging blood in which the hematocrit
level was regulated. This is because a hematocrit range of
approximately 30% was found to be the optimal value at which cerebral
oxygen delivery reaches a maximal peak during
hemodilution.14 18 Arterial blood samples were
taken for analysis of ATP and 2,3-DPG concentrations as well as
for measurement of hematocrit before and after transfusion.
Preparation of the Ischemic Rat Brain
Rats were further divided into three groups as follows:
ischemic 2,3-DPG–enriched RBC group, ischemic
2,3-DPG–subnormal RBC group, and nonischemic control group. In
the ischemic rat groups both common carotid arteries,
previously exposed through ventral midline cervical incisions, were
ligated 30 minutes after the exchange transfusion according to the
procedure of Fujishima et al.19 A small burr hole was made
in the right parietal bone, and cortical blood flow was measured by a
laser Doppler flowmeter (BPM 403, TSI). Sixty minutes after carotid
ligation the brain was frozen in situ by pouring liquid nitrogen into a
plastic funnel that was placed over the skull. The frozen brains were
then chiseled out carefully, and supratentorial
brain tissue was prepared for analysis.20 The
nonischemic control rat group was prepared in a similar manner;
that is, both common carotid arteries were exposed but not ligated
(sham operation). The brains in these rats not undergoing carotid
ligation were frozen 90 minutes after the exchange transfusion of blood
in which the RBCs were treated with either PEP or Pi.
Samples for Analysis
Blood samples were deproteinized immediately with ice-cold 0.6
mol/L HClO4. After standing for 5 minutes in ice, the
mixture of blood and perchloric acid was centrifuged at
16 000g for 10 minutes at 4°C. The extract was
neutralized with 5 mol/L KOH and used for analysis of ATP and
2,3-DPG contents.21 22 A 0.05-mL aliquot of blood was used
for measurement of hematocrit with the microhematocrit method by
centrifugation at 11 000 rpm for 5 minutes. The oxygen
dissociation curve and P50 of blood were determined
(Hemox-Analyzer, Technical Consulting Service) at pH 7.4 and
37°C. The frozen brain was weighed, powdered in liquid nitrogen, and
extracted for 10 minutes with 20 vol of 0.6 mol/L HClO4 at
0°C. The perchloric acid extracts were centrifuged at
24 000g for 10 minutes at 4°C, and the supernatants were
neutralized with 5 mol/L KOH before being used for the analysis
of adenine nucleotides, phosphocreatine, and
lactate.21 22
Materials
Both Wistar and spontaneously hypertensive rats were obtained
from Kyudo Co, Ltd (Kumamoto, Japan). All enzymes were obtained from CF
Boehringer und Soehne. Other reagents were of analytical
grade.
Statistics
All data were analyzed with the Statistical
Analysis System (SAS) computer program. All values are reported
as mean±SD; Student's t test was used for determination of
statistical significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Exchange Transfusion with 2,3-DPG–Enriched or –Subnormal RBCs
The 2,3-DPG content in rat RBCs increased from 6.06±1.41 to 13.61±4.24 µmol/mL cells after incubation with PEP, whereas it decreased from 5.71±0.64 to 2.94±0.73 µmol/mL cells after incubation with Pi. The ATP content in rat RBCs after PEP incubation also increased from 0.99±0.18 to 1.55±0.52 µmol/mL cells. No significant change in the ATP content was observed after incubation with Pi. There were significant differences in the RBC 2,3-DPG and ATP contents between the PEP- and Pi-treated groups (P<.001 and P<.05, respectively) (Table 1
). The
P50 of rat RBCs treated with PEP was also shifted from
32.0±1.0 to 46.7±4.7 mm Hg (n=3), as shown previously in both human
and canine RBCs.23 24 The 2,3-DPG content increased from
5.18±1.27 to 10.96±2.99 µmol/mL cells after the exchange
transfusion of blood in which RBCs were treated with PEP
(2,3-DPG–enriched RBC group). On the other hand, the 2,3-DPG content
decreased slightly from 4.99±0.94 to 3.77±1.06 µmol/mL cells after
the transfusion of blood in which RBCs were treated with Pi
(2,3-DPG–subnormal RBC group). There was a significant difference in
the RBC 2,3-DPG content between the 2,3-DPG–enriched and –subnormal
RBC groups after the transfusion (P<.001, Fig 1A). The ATP content increased from 0.74±0.11 to
0.99±0.11 and from 0.71±0.09 to 0.93±0.12 µmol/mL cells in both
the 2,3-DPG–enriched and –subnormal RBC groups, respectively. The ATP
content did not differ significantly between the two groups after
transfusion (Fig 1B).
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Changes in Brain ATP, Phosphocreatine, and Lactate Contents and
Cortical Blood Flow After Ligation
Fig 2 compares the relationships between hematocrit
and brain tissue metabolites after ischemia in the three
groups. When hematocrit was approximately 30% the ischemic
brain ATP content in both the 2,3-DPG–enriched and –subnormal RBC
groups ranged from 1.64 to 1.96 and from 1.60 to 1.63 µmol/g tissue,
respectively. They were preserved at 80% of the ATP level in the
nonischemic control group. As hematocrit was reduced to less
than 25% the ischemic brain ATP content remarkably decreased
in the 2,3-DPG–subnormal RBC group (Fig 2A, open triangles). However,
a slight change in the ischemic brain ATP content was also
observed in the 2,3-DPG–enriched RBC group under the same conditions
(Fig 2A, solid circles). The brain ATP content in the
nonischemic control group was maintained at 1.77 to 2.22
µmol/g tissue, with hematocrit values between 20% and 30% (Fig 2A,
open circles). The ischemic brain phosphocreatine contents in
the 2,3-DPG–enriched RBC groups were kept at 3.07 to 3.47 µmol/g
tissue, with hematocrit values from 20% to 30% (Fig 2B, solid
circles). However, in the 2,3-DPG–subnormal RBC group the
ischemic brain phosphocreatine content ranged from 2.48 to 2.96
µmol/g tissue and was already slightly different from the
ischemic brain phosphocreatine content in the two groups at a
30% hematocrit level. As hematocrit was reduced to less than 25% the
ischemic brain phosphocreatine content decreased to 1.50
µmol/g tissue in the 2,3-DPG–subnormal RBC group (Fig 2B, open
triangles). The brain phosphocreatine content in the
nonischemic control group was maintained at 3.33 to 3.80
µmol/g tissue, with hematocrit values ranging from 20% to 30% (Fig 2B, open circles). When hematocrit was approximately 30%, the
ischemic brain lactate content in both the 2,3-DPG–enriched
and –subnormal RBC groups ranged from 3.58 to 5.64 and from 2.55 to
3.69 µmol/g tissue, respectively. Their values were slightly higher
than those observed at the nonischemic control level. As
hematocrit was reduced to less than 25% the ischemic brain
lactate content remarkably increased in the 2,3-DPG–subnormal RBC
group (Fig 2C, open triangles) although a slight increase in the
2,3-DPG–enriched RBC group was observed under the same conditions (Fig 2C, solid circles). The brain lactate content in the
nonischemic control group was maintained at 1.29 to 1.97
µmol/g tissue, with hematocrit values between 20% and 30% (Fig 2C,
open circles).
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) or
2,3-bisphosphoglycerate–subnormal red blood cells (
) and brain
tissue metabolites after bilateral ligation of the common carotid
arteries (ischemia: 
). At 60 minutes after brain
ischemia, the heads of rats were frozen in situ with liquid
nitrogen, and the brains were carefully removed. ATP (A),
phosphocreatine (B), and lactate (C) were quantified as described in
"Materials and Methods." Each point indicates one individual
experiment.
Cortical blood flow in both the 2,3-DPG–enriched and –subnormal RBC
groups was reduced to 49.4±7.1% and 48.5±8.7% of the
preischemic control level, respectively, and there was no
significant difference in the changes between the two groups (Fig 3).
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Before and after ischemia, blood
PaCO2 and PaO2 as
well as arterial pH were all kept within normal ranges.
Mean arterial blood pressure increased by 20 to 25 mm Hg
60 minutes after ligation (Table 2). These results were
essentially similar to those of Fujishima et al.19 In
addition, there was no significant difference in these
parameters between the 2,3-DPG–enriched and –subnormal
RBC groups.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Since 2,3-DPG binding to hemoglobin and its regulative effect on blood oxygen-delivering capacity was first observed,4 5 the important role of RBCs in the oxygen dissociation capacity has been pointed out under both physiological and pathological conditions.11 25 26 27 28 RBCs with relatively high 2,3-DPG levels are generally more effective in oxygen delivery than 2,3-DPG–depleted RBCs. However, the advantage of RBCs having high 2,3-DPG in various pathological conditions, such as anemia, hypoxia, and ischemia, still remains to be elucidated. Several studies have described the effect of oxygen affinity in RBCs on cardiac function,11 25 but little has been reported on how the 2,3-DPG–enriched RBC affects tissue metabolism in the body.27 Several authors have found that the 2,3-DPG level in RBCs increased in either hypoxic or anemic conditions and thus emphasized that the increase of 2,3-DPG compensated for the lack of oxygen supply to the tissues.8 26 In contrast, however, it is hypothesized that a hypoxic condition causes gaseous alkalosis followed by an increase in RBC 2,3-DPG content, and the influence of an increase in the amount of RBC 2,3-DPG disappears because of Bohr's effect.9 10 We therefore designed the present study to investigate the influence of an increased RBC 2,3-DPG content on ischemic brain metabolism.
We demonstrated previously that extracellular PEP could penetrate the
RBC membrane and be metabolized to 2,3-DPG and ATP accompanied by an
increase in P50.16 17 23 29 When canine RBCs
treated with PEP were autologously transfused, the elevated 2,3-DPG and
P50 values in the circulating blood were maintained during
the second day after the transfusion.24 Essentially the
same results were observed in open-heart surgery
patients.30 In the present study the 2,3-DPG
concentration in rat RBCs increased by 220% as a result of PEP
treatment in vitro, as shown previously in both human and canine RBCs
(Table 1
).16 23 24 The circulating blood 2,3-DPG
concentration increased to 200% of the pretransfusion level when these
cells were transfused (Fig 1). The RBC oxygen-delivering capacity
increases as a reflection of 2,3-DPG increase in the cells. The
increased level of 2,3-DPG to 200% of normal value in rats exchanged
with the 2,3-DPG–enriched RBCs resulted in an accelerated
oxygen-delivering activity that was approximately 175% that of normal
rats.31
Cerebral blood flow increases as hematocrit decreases, and this action forms the theoretical foundation of hemodilution therapy for cerebral ischemia.12 13 However, this increased blood flow does not necessarily improve the oxygen transport and oxygenation of cerebral tissue because it may be caused by the balance between the reduced arterial oxygen content and increased cerebral blood flow.12 14 15 18 In fact, the optimal value to which hematocrit should be reduced for the best compromise to be achieved in the improvement of cerebral blood flow and maintenance of oxygen delivery has not yet been satisfactorily determined. Wood et al15 demonstrated a favorable inverse relationship between regional cortical oxygen transport and hematocrit in ischemic brain, and Kiyohara et al14 also found that there is a significant inverse U-shaped correlation between hematocrit and ATP content in ischemic brain, with a maximal hematocrit level of 37%. The optimal hematocrit value for brain ischemia has been considered to be just above 30%.13 18
In the present study the ischemic brain ATP,
phosphocreatine, and lactate contents did not differ between the
2,3-DPG–enriched and –subnormal RBC groups at a hematocrit value of
30% (Fig 2), indicating that this value is approximately optimal for
ischemic brain tissue oxygenation in the
2,3-DPG–subnormal RBC group. However, when hematocrit was reduced to
less than 25% the ischemic brain ATP and phosphocreatine
contents remarkably decreased and the lactate content substantially
increased in the 2,3-DPG–subnormal RBC group. In contrast, in the
2,3-DPG–enriched RBC group ischemic brain ATP and
phosphocreatine contents were kept as high as those in the
nonischemic group, and the lactate content increased moderately
under the same conditions (Fig 2), indicating that the impairment of
ischemic brain metabolism caused by the
insufficient oxygen supply was compensated with the high
oxygen-delivering capacity of the 2,3-DPG–enriched RBCs. The decrease
of phosphocreatine preceded that of ATP in the 2,3-DPG–subnormal group
(Fig 2A and 2B), consistent with previous
results.32
Cortical blood flow decreased to 10% of preischemic levels
60 minutes after bilateral carotid ligation in spontaneously
hypertensive rats in which hematocrit was normal.19 33 In
the present study the degree of reduction in cortical blood flow
remained at 50%, with hematocrit ranging from 20 to 30% (Fig 3). This
indicates that hemodilution, during which hematocrit values were
reduced from 50% to 20-30%, increases cerebral blood flow during
brain ischemia. Cortical blood flow did not differ
significantly between the 2,3-DPG–enriched and –subnormal RBC groups
(Fig 3).
We could conclude from these data that the ischemic brain metabolism of the 2,3-DPG–enriched RBC group was kept as normal as that of the nonischemic group, whereas that of the 2,3-DPG–subnormal RBC group deteriorated, because 2,3-DPG–enriched RBCs had a high oxygen-delivering capacity and thus could compensate for the insufficient oxygen supply in extremely anemic conditions. These results also suggest that 2,3-DPG–enriched RBCs modified by PEP may be clinically useful during hemodilution therapy in stroke patients.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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We would like to thank Prof Sheshadri Narayanan (New York Medical College) for critical reading of the manuscript.
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Footnotes |
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Reprint requests to Hideo Kimura, MD, Department of Neurosurgery, Fukuoka University School of Medicine, 7-45-1 Nanakuma Jounan-ku, Fukuoka 814-01, Japan.
Received May 23, 1994; revision received March 28, 1995; accepted April 20, 1995.
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