| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Department of Polymer Chemistry,*
Advanced
Research Institute for Science and Engineering, Waseda University,
Tokyo; and the Departments of Surgery
and Pathology,
Keio University School of
Medicine, Tokyo, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
250 nm) most
closely mimics the characteristics of natural red blood cells such as
the cell membrane function of physically preventing direct contact of
Hb with the components of blood and vasculature during circulation. In
comparison with some acellular Hb modifications, Hb encapsulation in
vesicles not only prolongs the circulation time, but also suppresses
hypertension induced by vasoconstriction; a theory that is suggested to
be due to the high affinity of Hb with nitric oxide and carbon monoxide
as vasorelaxation factors.11-13 Free Hb released from RBC is rapidly bound to haptoglobin and removed from the circulation by hepatocytes. However, when the Hb concentration exceeds the haptoglobin binding capacity, unbound Hb is filtered through the kidney where it is actively absorbed. When the reabsorption capacity of the kidney is exceeded, hemoglobinuria and eventually renal failure occur.14 The encapsulation of Hb completely suppresses renal excretion, though HbV particles as well as phospholipid vesicles (liposomes) or oil emulsions in the blood stream are finally captured by phagocytes in the reticuloendothelial system (RES, or mononuclear phagocytic system, MPS).15-17 A chemically modified (pyridoxalated) Hb was also reported to be captured by RES.18 The main function of RES is protective elimination of foreign materials such as viruses, bacteria, metastatic tumor cells, and bacterial endotoxins.19 It has been anticipated that the accumulation of phospholipid vesicles in phagocytic cells may lead to impairment or even blockade of their function.20 This may cause adverse effects such as decreased resistance to infection, the metastatic spread of tumors, or spillover of endotoxins from the gut. Clinically approved Fluosol-DA (Green Cross Co., Osaka, Japan) as an oxygen carrier and lipid emulsion as a nutrient are trapped by the phagocytic cells.21-24 Several reports measured the phagocytic activity by the carbon clearance method and clarified that the phagocytic activity is transiently depressed after the infusion and then recovered and enhanced.25-28
The advantages of our HbV over the conventional Hb vesicles29-31 are a high encapsulation efficiency of Hb in phospholipid vesicles thus the total amount of lipids is significantly reduced,32-33 the surface modification of HbV with poly(ethylene glycol) that allows better hemodynamics due to the suppression of intervesicular aggregation and reduced viscosity,34-36 reduced complement activation due to the suppression of interaction with plasma proteins,37 and longer circulation time and a moderate rate of entrapment and metabolism.38 We thought these revised characteristics may be effective to maintain microcirculation and to reduce the burden on RES. In this study, the effect of HbV infusion on the RES function was analyzed by the carbon clearance measurement,25,28,39 and also its metabolism and the influence on the tissue parenchymal cells was confirmed by histopathological examination.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Injection of Sample Suspensions
All animal studies were approved by the Animal Subject Committee of Keio University School of Medicine and performed according to NIH guidelines for the care and use of laboratory animals (NIH publication 85–23 Rev. 1985).
Experiments were carried out using 70 male Wistar rats (200–210 g, Charles River Co., Tokyo, Japan). They were anesthetized with diethylether, and the sample suspension was infused into the tail vein. The sample was either HbV (10 ml/kg, n = 15; 20 ml/kg, n = 19) or saline (20 ml/kg, n = 15) and 10 wt% of IntralipidTM suspension (Pharmacia, Stockholm, Sweden) 20 ml/kg, n = 15). Six animals were used to obtain the control values. All of the rats were housed in cages and provided with food and water ad libitum in a temperature controlled room on a 12 hour dark/light cycle.
Carbon Clearance Measurement
After 8 hours and 1, 3, 7, and 14 days, the rats were anesthetized
with an intraperitoneal injection of sodium pentobarbital ( 100
mg/kg body weight, Abbott Lab., North Chicago, IL). Polyethylene tubes
(PE-50, Natsume Co., Tokyo) were implanted in the jugular vein. A
carbon particle solution (Fount India Ink, Pelikan Co., Hannover,
Germany) was diluted to 16 mg/ml with saline and infused at 10 ml/kg
within 1 minute. The pink-colored rat skin immediately turned to black,
indicating that the carbon particles were circulating throughout the
body. Four, 10, and 20 minutes later, about 120 µl of blood was
withdrawn from the vein, and exactly 50 µl of blood was diluted with
5 ml of a 0.1% sodium bicarbonate solution in a cuvette for
spectrophotometer. Absorption at 675 nm was measured with the
spectrophotometer (UV-2000, Shimadzu Co., Tokyo, Japan). The control
blood was also measured before infusing the carbon particle solution.
The phagocyte index (K) was calculated with the
equation: K =
1/(t2-t1)
x
ln(C1/C2)
where C1 and
C2 are the concentrations (absorbance)
at time t1 and
t2 (minutes), respectively. After the
experiment, the animals were laparotomized to be sacrificed with acute
bleeding from the abdominal aorta and to obtain the liver, spleen, and
kidney, and then the lung and heart were resected en bloc
for a histopathological study. The organs were soaked in 10% formalin
immediately after the resection.
Histopathological Study
Paraffin sections were prepared from the 10% formalin-fixed organs, and stained with hematoxylin/eosin, anti-human Hb antibody, Berlin blue, and oil red O stainings. The human Hb in the HbV particles in the tissue was confirmed by staining with a rabbit polyclonal antibody against human Hb (DAKO A/S, Copenhagen, Denmark) as the primary antibody. This antibody does not cross-react with rat hemoglobin (which was evident from the result that rat red blood cells were not stained). Reaction with the second antibody and color development were performed with the Ventana alkaline phosphatase RED detection kit using the Ventana NX system (Ventana Med. System, Inc., Tucson, AZ). The percentage of the stained area was calculated with a computer software (IPLab, Fairfax, VA). The presence and location of hemosiderin including free irons released by the metabolism of heme were confirmed by Berlin blue stain. The neutral lipid deposition, which might be generated during the metabolism of the phospholipid components of the bilayer membrane of HbV, were examined by oil red O staining of the sliced organ specimens directly prepared from the formalin-fixed organs.
To visualize the morphological changes in the HbV particles in the spleen and liver, transmission electron microscopic observation (with a high magnification) was performed. The spleen and liver, taken from the rat without carbon particle infusion, were cut in about 2 mm3 portions in 2.5% glutaraldehyde solution and then stored in 8% sucrose solution (0.1 mol/L phosphate buffer, pH 7.4). The fixed organs were then washed with 0.1 mol/L phosphate buffer, and stained with 2% osumic acid solution at 4°C for 2 hours. Next, the organs were first dehydrated with ethanol solution by stepwise increases in the ethanol content (50, 60, 70, 80, 90, 95, and 100%) for 10 minutes during each step, washed with propylene oxide, and then polymerized using Quetol 812 at 60°C for 28 hours. The obtained samples were sliced into 60 to70-nm sections by using an Ultracut S microtome. The sliced samples were stained with 3% uranyl acetate solution for 16 to 20 minutes and then treated with Satoh’s lead solution (lead acetate, lead nitrate, and lead citrate) in citrate for 5 minutes, washed, and dried. The sample was observed and a picture taken with a transmission electron microscope (TEM, JEM-100CX, JEOL, Tokyo, Japan).
Blood Serum Clinical Chemistry
The rats receiving HbV but not carbon particles were used to analyze blood serum clinical chemistry (n = 15). After 8 hours and 1, 3, 7 and 14 days, the rats were anesthetized with an intraperitoneal injection of sodium pentobarbital. Polyethylene tubes (PE-50) were implanted in the carotid artery and about 4 ml of blood was withdrawn in heparinized syringe. Since HbV particles interfere with some analytes of serum clinical chemistry, the blood was ultracentrifuged (50,000 x g for 20 minutes) to completely remove the HbV particles in advance. The serum samples were stored in a refrigerator (-80°C) until the analyses. To evaluate function of liver as one of the main organs for the HbV metabolism, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were selected as the analytes (BML Inc., Kawagoe, Japan).
Data Analysis
Differences between the treatment groups were analyzed using a one-way analysis of variance followed by Fisher’s protected least significant difference (PLSD) test. A paired t-test was used to compare the time dependent changes within each group. The changes were considered statistically significant if P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
All of the rats tolerated the overdose of the sample solutions.
The original body weights were 210 to 225 g on average (Figure 1a)
. The body weight decreased,
especially in the 20 ml/kg HbV group, by about 10% one day after the
HbV administration. However, the body weight of the HbV groups returned
to that of the control groups within 3 days and they grew normally.
|
. The spleen weight
increased for the 20 ml/kg HbV group by about 500 mg, which is about
70% of the infused amount of HbV (3300 mg/kg x 0.21 kg =
693 mg), and the spleen weight tended to remain even after 2 weeks. The
10 ml/kg HbV group showed a spleen weight increase at 1 week and tended
to decrease at 2 weeks. Changes in Phagocytic Activity
The phagocytic index (K) dropped at 8 hours or 1
day after the HbV infusion by about 30 to 50%, though it never
completely saturated (Figure 2)
. In the
case of the 10 ml/kg HbV group, the K value recovered 3 days
later and showed a maximum value at 1 week, and then ceased at 2 weeks.
For the 20 ml/kg HbV infusion, the significantly high value of
K, about twice the baseline value, was observed at 1 week,
and then it ceased at 2 weeks. Thus the dramatic changes in the
phagocytic activities were not irreversible. The changes in
K for the saline and lipid microsphere groups were minimal.
|
The histopathological examination of the spleen and liver after
the 20 ml/kg infusion is shown in Figure 3
. The human Hb in HbV particles were
stained as red-colored portions with anti-human Hb antibody as a
primary antibody. It was confirmed in advance with smears of human and
rat blood and HbV suspension that the antibody reacts with only human
Hb but not with rat Hb.
|
. A significant amount of macrophages
in spleen entrapping HbV particles was seen in the red pulp zone as
red-colored domains (Figure 3, a, c, and e)
. The total area of the
red-colored portion in the red pulp zone was 31.0 ± 6.1% at one
day, then it gradually decreased to 5.1 ± 2.0% after 3 days, and
to less than 0.05% after 7 days (Figure 4)
. On the other hand, a significant
amount of carbon particles was seen in the marginal zone around the
white pulp, where lymph cells are located, indicating the enhanced
phagocytic activity (Figure 3, a, c, and e)
. For the liver, Kupffer
cells trapping HbV particles were seen as a red-stained area at one day
after the infusion (Figure 3b)
. After 3 days, the HbV as well as a
large amount of carbons were seen in the same position (Figure 3d)
. The
HbV particles completely disappeared 7 days after infusion while the
Kupffer cells trapping a large amount of carbon were observed which
corresponded to the enhanced phagocytosis (Figure 3f)
. The total area
of the red-colored portion was 7.6 ± 1.9% at one day, then it
gradually decreased to 1.3 ± 0.2% after 3 days, and almost
completely disappeared after 7 days (Figure 4)
.
|
. However, at 14 days,
no stain was confirmed either in the spleen or liver, indicating that
the heme metabolism from Hb proceeded smoothly.
|
. The dye,
oil red O, locates in the domain of neutral lipid (e.g., triglyceride).
Therefore, this result indicated that the metabolism of phospholipid
components proceeded smoothly, and there was no deposition of the
metabolites. In kidney and lung no significant pathological infarcts,
such as capillary emboli with HbV particles, are noted in either organ
(Figure 7, a and b)
.
|
|
Transmission electron microscopy (TEM) observations of the
phagocytic cells clearly demonstrated the presence of HbV particles in
the phagosomes and capillaries both in the liver and spleen after 1 day
(Figure 8, a and b)
. The HbV diameter is
about 250 nm which is about 1/40 of red blood cells. Since one Hb
contains four irons and a resulting higher electron density, the
Hb-vesicles as well as red blood cells are black in color. Some HbV are
seen near the RBCs indicating that they are in capillaries and most of
the HbV are captured by the phagocytic cells. After 3 days, HbV cannot
be seen in the capillaries and the HbV is mostly captured by the RES.
After 7 days, no phagosomes with HbV particles can be observed (Figure 8, c and d)
.
|
The AST and ALT values before the infusion of HbV were 65.4 ± 2.7 units/l and 29. 6 ± 3.5 units/l, respectively. After the bolus infusion of HbV at 20 ml/kg dose rate, they significantly increased to 483 ± 85 units/l and 149 ± 5 units/l at 8 hours, respectively. However, after 3 days they returned to the original levels; AST was 47.5 ± 7.8 units/l and ALT was 44.0 ± 15.5 units/l. The values were almost unchanged until 14 days after the infusion.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The carbon clearance measurement showed that the systemic phagocytic activity decreased by 40% soon after the HbV infusion. Since the circulation time of HbV at the used condition is about 20 to 30 hours, the peak of the HbV accumulation is estimated to be around 1 to 2 days after the infusion, and this corresponds to the significantly decreased phagocytic activity at 8 hours and 1 day. However, it recovered 3 days after the infusion, and then it increased by 200%. The same tendency of suppression and the succeeding enhancement of phagocytic activity was reported for other oxygen carriers, i.e., Fluosol-DA28 and liposomes for cancer therapy.25 The influence of the transient decrease in phagocytic activity after the infusion of HbV should be investigated in detail, especially in the septic shock model when the defense system in a body is significantly depressed.16,45 However, the phagocytic activity returned to the original level at 2 weeks. The results of the serum clinical chemistry (AST and ALT) indicate that the level of liver function decreased temporarily and recovered to the normal level within 3 days. Judging from these results, the change of liver was considered transient and functional. The initial body weight loss, especially the 20 ml/kg HbV group one day after the HbV administration, may be related to the decreased appetite since the animals did not fast from food and water. However, the body weight of the HbV groups returned to that of the control groups within 3 days and they grew normally.
In our study, simultaneous splenomegaly was observed for the HbV high
dose group 3 days after infusion which corresponded to the decreased
plasma concentration of HbV (half life of HbV: 2030 hours). However,
the spleen weight did not return to the control value in 2 weeks
despite the fact that the phagocytic activity returned to the control
value. Histopathological examination indicated that the HbV particles
were metabolized within one week, and there was no infarcts in the
spleen, indicating that the sustained splenomegaly is not due to the
remaining non-metabolized HbV elements but due to the increased amount
of phagocytic or parenchymal cells and/or red blood cells.
The administration of lipid microspheres (IntralipidTM) did not greatly influence the phagocytic activity and spleen weight. We selected the lipid microspheres as a control injection according to some reports which showed that lipid microspheres suppress the RES function.21,24,46,47 The reasons for the small amount of change should be that the total concentration of solutes in a lipid emulsion (10%) is lower than that in the HbV suspension (16 wt %), and that the metabolic route of the HbV is different from that of the lipid microspheres. It was reported that the lipid emulsions were mainly consumed by muscle and fat, with a negligible contribution from the liver and spleen.48,49
The histopathological examination clearly demonstrated that the HbV accumulated in the liver was localized in the Kupffer cells. Hepatocytes appeared normal and lobular architecture was not affected by the HbV administration. This accumulation subsided gradually during the experimental period. The HbV accumulated in spleen was localized in macrophages in the red pulp and marginal zone. Uptake of HbV in the spleen may occur using a filtration mechanism50 and opsonization.51 There was no aggregation of HbV particles in renal and pulmonary capillaries at any time. Rudolph et al52 indicated small pulmonary and glomerular infarcts due to the trapping of large clusters of aggregated particles of the liposome-encapsulated Hb. The results of this study reflect the differences in the physicochemical characteristics of the vesicles. The particle diameter of our HbV is well-regulated to about 250 nm by the extrusion method, and the vesicular surface is modified with PEG to suppress the intervesicular aggregation34 and this may effectively prevent embolization of capillaries and venules in microcirculation.35, 36
Transmission electron microscopy (TEM) one day after infusion revealed that the HbV particles were captured in the macrophages in the spleen and Kupffer cells by endocytosis and not by fusion because the intact HbV particles were present in the phagosomes. The presence of HbV near the RBC indicates the circulating HbV in vasculature. The vesicular structure of HbV completely disappeared at 7 days indicating that they are totally destroyed in the phagosomes. TEM was a very effective tool for detecting the HbV particles in tissues. The conventional method to detect liposomes (phospholipid vesicles) required encapsulation of colloid gold as a microscopic marker.53, 54 On the other hand, the high electron density of HbV due to the highly concentrated Hb solution in the inner aqueous phase of HbV as well as in RBC provided sufficient contrast of the particle and clearer images than those displayed in other reports to our knowledge.
Anti-human Hb antibody staining was very effective for staining the human Hb-based oxygen carriers in animal tests. The antibody recognizes the globin chain of human Hb in HbV and there is no cross-reaction with rat Hb. The stained portion mostly disappeared within 7 days both in the spleen and liver. The released heme from Hb in HbV may probably be metabolized by the inducible form of heme oxygenase-1 in the Kupffer cells in the liver 13 and in the spleen.55 Normally, iron from a heme is stored in the ferritin molecule.56 This protein has 24 subunits and encloses as many as 4500 iron atoms in the form of an aggregate of ferric hydroxide. Ferritin in the lysosomal membrane may form paracrystalline structures and eventually aggregate in mass with an iron content as high as 50%. These are hemosiderins composed of degraded protein and coalesced iron. It was reported that polymerized Hb were captured by the Kupffer cells and then hemosiderin was confirmed, and the renal tubules showed siderosis.14 On the other hand, in our HbV study, there were slight Berlin blue stains only in spleen and liver after 3 and 7 days, and they completely disappeared after 14 days. As the anti-human Hb antibody staining disappeared, trace staining of hemosiderin was recognized and it completely disappeared after 14 days. This indicates that the amount of the stored hemosiderin was minimal, thus the metabolism of heme and recycling of iron molecule are suggested to be on the physiological pathway.
As for the membrane components of HbV, it was reported that the infused lipid components of liposomes are entrapped in the Kupffer cells, and diacylphosphatidylcholine is metabolized and reused as a component of the cell membrane, or excreted in bile, especially as fatty acids’ and in exhaled air.57-59 Liposomal cholesterol reappears in blood as lipoprotein cholesterol after entrapment in the Kupffer cells, and then is excreted in bile after entrapment of the corresponding lipoprotein by the parenchymal hepatocytes.60 Even though the amount of the infused HbV is much greater than in these studies, the main vesicular components of HbV, the phospholipids and cholesterol, would gradually be metabolized in the same manner. There were small amounts of trace staining with oil red O only in liver 3 days after infusion. This indicated that the deposition of neutral lipid components such as glycerides, which may be produced during the metabolism, was minimal. However, further study is necessary to clarify the influence of a large dose of HbV, especially on a lipemic model.
The long-term stability of HbV during storage has been a major issue for the practical use of oxygen carriers especially for emergency situations. In the previous reports the polymerization of phospholipids bearing dienoyl groups in the bilayer membrane of HbV has shown the enormous stabilization of HbV against long term storage in a freezer or as a freeze-dried powder.61-63 However, the polymerized phospholipid is difficult to decompose or metabolize and the bilayer structure remained even after 30 days with showing cytotoxicity.64 After infusing polymerized phospholipid, the phagocytic activity remained suppressed even after 3 days, and the recovery was observed after 7 days.26 On the other hand, in our method, the storage stability of our HbV is achieved by the surface modification with PEG chains and deoxygenation.6 Accordingly, our PEG-modified HbV shows rapid metabolism once trapped by RES. The PEG-lipid is susceptible to hydrolysis to release PEG chains during metabolism. The released PEG chains, which is known as an inert macromolecule, should be excreted in the urine through the kidneys.65 Our HbV possesses a high encapsulation efficiency of Hb, so that the weight ratio of Hb to the total lipid component is nearly 1.6 to 1.8. This is much higher than the previously reported liposome encapsulated Hb. The reduced amount of lipid is beneficial for reducing the burden on RES. Even though several groups have tried so called liposome encapsulated Hb without polymerized phospholipid, there are few reports on the metabolism and the fate of the components. Sakaguchi et al27 tested the metabolism of their product, Neo Red Cells, histopathologically with the whole body autoradiograms. However, the concentration of Hb and infusion amount were lower than our case. Therefore, our study demonstrates more detailed information with a larger dose rate of HbV for a longer period of observation.
In conclusion, the components of the infused HbV (20 ml/kg i.v.) entrapped in the RES were smoothly metabolized within one week. Even though the HbV infusion modified the phagocytic activity for two weeks, it does not seem to cause any irreversible damage to the phagocytic organs. Our results are demonstrated only in healthy rats, while rats in hemorrhagic shock or septic shock, or with an inflammation reaction, may react differently in these pathological situations. The infused amount in this study is equal to about 1.4 l in a 70-kg man and larger volume is needed for the further evaluation. Hence safety concerns cannot be totally eliminated by this study. However, our results offer important information for evaluating the safety issues of HbV for clinical use.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported in part by Health Sciences Research Grants (Research on Advanced Medical Technology, Artificial Blood Project), the Ministry of Health, Labour, and Welfare, Japan (12090101), Grants in Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (12480268, 12558112), and Grants from the Mukai Science and Technology Foundation.
Accepted for publication June 12, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-ray polymerization. Polymers Adv Technol 1992, 3:389-394
This article has been cited by other articles:
 |
|
 |
|
  K. Taguchi, Y. Urata, M. Anraku, T. Maruyama, H. Watanabe, H. Sakai, H. Horinouchi, K. Kobayashi, E. Tsuchida, T. Kai, et al. Pharmacokinetic Study of Enclosed Hemoglobin and Outer Lipid Component after the Administration of Hemoglobin Vesicles as an Artificial Oxygen Carrier Drug Metab. Dispos., July 1, 2009; 37(7): 1456 - 1463. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Awasthi, S.-H. Yee, P. Jerabek, B. Goins, and W. T. Phillips Cerebral oxygen delivery by liposome-encapsulated hemoglobin: a positron-emission tomographic evaluation in a rat model of hemorrhagic shock J Appl Physiol, July 1, 2007; 103(1): 28 - 38. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Yamazaki, R. Aeba, R. Yozu, and K. Kobayashi Use of Hemoglobin Vesicles During Cardiopulmonary Bypass Priming Prevents Neurocognitive Decline in Rats Circulation, July 4, 2006; 114(1_suppl): I-220 - I-225. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Sakai, P. Cabrales, A. G. Tsai, E. Tsuchida, and M. Intaglietta Oxygen release from low and normal P50 Hb vesicles in transiently occluded arterioles of the hamster window model Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H2897 - H2903. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Matsumoto, T. Asano, K. Mano, H. Tachibana, M. Todoh, M. Tanaka, and F. Kajiya Regional myocardial perfusion under exchange transfusion with liposomal hemoglobin: in vivo and in vitro studies using rat hearts Am J Physiol Heart Circ Physiol, April 1, 2005; 288(4): H1909 - H1914. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Sou, R. Klipper, B. Goins, E. Tsuchida, and W. T. Phillips Circulation Kinetics and Organ Distribution of Hb-Vesicles Developed as a Red Blood Cell Substitute J. Pharmacol. Exp. Ther., February 1, 2005; 312(2): 702 - 709. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Sakai, Y. Masada, H. Horinouchi, E. Ikeda, K. Sou, S. Takeoka, M. Suematsu, M. Takaori, K. Kobayashi, and E. Tsuchida Physiological Capacity of the Reticuloendothelial System for the Degradation of Hemoglobin Vesicles (Artificial Oxygen Carriers) after Massive Intravenous Doses by Daily Repeated Infusions for 14 Days J. Pharmacol. Exp. Ther., December 1, 2004; 311(3): 874 - 884. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Sakai, Y. Suzuki, M. Kinoshita, S. Takeoka, N. Maeda, and E. Tsuchida O2 release from Hb vesicles evaluated using an artificial, narrow O2-permeable tube: comparison with RBCs and acellular Hbs Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2543 - H2551. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Sakai, S. Takeoka, R. Wettstein, A. G. Tsai, M. Intaglietta, and E. Tsuchida Systemic and microvascular responses to hemorrhagic shock and resuscitation with Hb vesicles Am J Physiol Heart Circ Physiol, September 1, 2002; 283(3): H1191 - H1199. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
