CA1130724A

CA1130724A - Controlled improvement of the o.sub.2-release by intact erythrocytes

Info

Publication number: CA1130724A
Application number: CA310,662A
Authority: CA
Inventors: Yves-Claude Nicolau; Klaus Gersonde
Original assignee: Studiengesellschaft Kohle gGmbH
Current assignee: Studiengesellschaft Kohle gGmbH
Priority date: 1977-09-06
Filing date: 1978-09-05
Publication date: 1982-08-31
Also published as: IT1111675B; JPS6022683B2; AU524587B2; ES473087A1; YU208078A; JPS5449314A; IN149223B; ATA642478A; EP0001104A2; DE2860719D1; IE47329B1; EP0001104A3; AU3955178A; DK393878A; EP0001104B1; IT7827388A0; AT364086B; IE781800L

Abstract

BY INTACT ERYTHROCYTES

Abstract of the Disclosure The invention provides erythrocytes of improved O2-release properties having fused thereto small allosteric effectors, f.i. inositol hexaphosphate-containing lipid vesic-les. Also included in the invention is a method for making said erythrocytes and plasma containing said erythrocytes.

Description

lI~07Z~

One of the major functions of erythrocytes con-sists in the transport of molecular oxygen from the lungs to the peripheral tissues. The erythrocytes contain a high concentration of haemoglobin (Hb) (30 pg per cell = 35.5 g/100 ml cells) which forms a reversible adduct with 2 The 02-partial pressure in the lung is ~_100 mm Hg, in the capillary system ~70 mm Hg against which 2 must be dis-sociated from the oxygenated haemoglobin. Under physio-logical conditions only about 25% of the oxygenated haemo-globin may be deoxygenated;r-75% is carried back to the lungs with the venous blood. Thus, the major fraction of the Hb-O2 adduct is not used for the 2 transport.
Interactions of Hb with allosteric effectors enable an adaptation to the physiological requirement of maximum 2 release from the Hb-O2 adduct with simultaneous conservation of the highest possible O2-partial pressure in the capillary system.
Hb's allosteric properties are responsible for the sigmoid shape of the O2-binding curve. Its half-saturation pressure (PO (1/2) ) is an indicator of the 2affinity. The slope of the sigmoid curve in the range of ~10 to 60~ saturation represent~ the degree of cooper-ativity of the four O2-binding sites in Hb (Hill coeffi-cient: n = 2.8 - 3.2). An increase of the Hill coefficient, i.e. a steeper slope of the sigmoid binding curve, or a shift of the entire binding curve towards higher 2-partial pressure (so-called "right-shift") would lead to a racilitated 2 release in the capillary system and to an improved oxygen supply to the surrounding tissues.

- 2 -`,.'1 , ~307Z4 A "right-shift" is actually achieved in vivo by the enhance-ment of the 2,3-bisphosphoglycerate level in erythrocytes and its binding to haemoglobin. Binding of 2,3-bispho-sphoglycerate to Hb decreases the 2 affinity and increases the 2 half-saturation pressure. 2,3-bisphosphoglycerate synthesized in the glycolysis bypass in erythrocytes is an effector for slow adaptation to 2 deficiency.
2,3-bisphosphoglycerate increases the half-saturation pressure of stripped haemoglobin at pH 7.4 from PO (1/2) = 9.3 mm Hg (37C), and 4.3 mm Hg (25C) to PO (1/2) = 23.7 mm Hg (37C), and 12.0 mm Hg (25C), respectively (Imai, K. and Yonetani, T. (1975), J. Biol.
Chem. 250, 1093 - 1098). A significantly stronger decrease of the 2 affinity, i.e. enhancement of the 2 half-satur-ation pressure has been achieved for stripped haemoglobin by binding of inositol hexaphosphate (phytic acid; IHP) (Ruckpaul, K. et al. (1971) Biochim. Biophys. Acta 236, 211 - 221) isolated from vegetal tissues. Binding of IHP
to haemoglobin increases the 2 half-saturation pressure to PO (1/2) = 96.4 mm Hg (37C), and PO (1/2) = 48.4 mm HG
(25C), respectively. I~IP, like 2,3-bisphosphoglycerate and other polyphosphates cannot permeate the erythrocyte membrane.
Lipid vesicles reflect some of the properties of cell membranes. They can penetrate into cells either by fusion with the cell plasma membrane or by endocytosis (Papahadjopoulos, D. et al. (1976) Nature 252, 163 - 165).
The fusion plays an important part in a number of membrane processes like: Synaptic transmission, secretion, plasma membrane assembly, infection with enveloped viruses. The 1~30724 fusion of cells with lipid vesicles was achieved in cell cultures and with erythrocyte ghosts.
Treatment of ~L cells (cell line-murine lymphoma) with vesicles containing IgG with a high neutralizing titer against Coxsackie ~-irus A-2 L protected the cells against subsequent infection with this ~irus. Gregoriadis and Buckland (Nature (1973) 235, 252 - 253) have used lipid vesicles in order to incorporate invertase into invertase-defective mouse macrophages. In vivo experiments though, did not lead to unambiguous results since the lipid vesicles are incorporated indiscriminately by all cells or are quick-ly destroyed in the liver.
The use of lipid vesicles for the incorporation of allosteric effectors or of other substances into erythro-cytes has not been reported so far. Many experiments haveshown that lipid vesicles can be injected into animals with-out danger.
Whereas the influence of IHP on the structure and function of isolated Hb has been studied in detail there are no data about the interaction of I~P with hae-moglobin in intact erythrocytes, as it was not possible, until now, to incorporate IHP into intact erythrocytes.
Surprisingly, we found that using fluid-charged lipid vesicles, which can fuse with the erythrocyte mem-brane, it was possible to transport allosteric effectors2S IHP into erythrocytes where, due to its much higher binding constant it replaces 2,3-bisphosphoglycerate at its binding site in haemoglobin. Under these conditions F;b in erythrocytes change into an allosteric conformation with a significantly lower 2 affinity.

~130724 The lowering of the 2 affinity, i.e. the enhance-ment of the 2 half-saturation pressure of haemoglobin in erythrocytes, increases the capacity of erythrocytes to dissociate the bound 2 even against higher O2-partial pressures and thus improves the 2 supply to the tissues.
Erythrocytes, having incorporated IHP and which therefore provide an improved 2 supply to the tissues may find their use in the following cases:
1. Under low O2-partial pressure in the inhaled air:
Mountain climbers at high altitudes, astronautes in O2-poor atmosphere.
2. In the case of reduced O2-exchange surface:
Decrease of the number of pulmonary alveoles in lung emphysema.

3. Increased resistance to 2 diffusion in the lung:
Pneumonia, Asthma.

4. Decreased O2-transport capacity: Erythropeny, anaemic states of all types, arteriovenous shunt.

5. Blood circulation disturbances: Arteriosclerosis, thromboembolic processes, organ infarct, ischaemic states.

6. High 2 affinity of haemoglobin: Haemoglobin mutations, chemical modification of N-terminal amino acids in the haemoglobin-chains e.g.
Diabetes mellitus, enzyme defects in erythrocytes.

7. Acceleration of detoxication processes by improved oxygen supply.

8. In order to decrease the O2-affinity of conserved blood: Transfusion, shock states.

9. Improvement of radiotherapy of cancer.

As allosteric effectors ~or the incorporation into erythrocytes are used which have a larger affinity -o haemoglobin than the known physiologic effectors, 2,3-bisphosphoglycerate and adenosine triphosphate.
Specifically inositol hexaphosphate is preferred as allosteric effector.
Other sugar phosphates as inositol pentaphos-phate, inositol tetraphosphate, inositol triphosphate, inositol diphosphate and diphosphatidylinositol diphosphate can be used as allosteric effectors.
Further allosteric effectors are other poly-phosphates as nucleotide triphosphates, nucleotide diphos-phates, nucleotide monophosphates, and alcohol phosphate esters are suitable.
In case of certain mutations of haemoglobin, f.i.
"Zurich" haemoglobin, orgainc anions as polycarboxylic acids can be used as allosteric effectors.
Finally, it is possible to use inorganic anions as hexacyano ferrate, phosphate or chloride as allosteric effectors.
Lipid vesicles are mixtures of phosphatidyl-choline/phosphatidylserine/cholesterol which are used in a mole ratio of 10 to 5 : 4 to 1 : 10 to 3. Specificall~
favored is a mole ratio of 8 : 2 : 7, but also a mole ratio of 9 : 1 : 8 as well as 8 : 4 : 7 is favorable.
As fluid carrier the customary liquid carrier, specifically buffered physiologic carriers are used which are ~nown, f.i. isotonic bis-Tris buffer.
The lipid vesicles must be able to fuse with the membrane of the erythrocytes. By the incorporation ~, of allosteric effectors into intact erythrocytes it is pos-si~le now that haemoglobin of the erythrocytes is trans-ferred into an allosteric conformation which releases oxygen easier.
`By the invention it is possible to produce modi-fied erythrocytes which guarantee an improved oxygen economy of the blood. These modified erythrocytes are obtained by irreversible incorporation of allosteric effectors which takes place by fusing lipid vesicles into the erythrocyte membranes and binding the allosteric effectors so introduced into the erythrocytes to the haemoglobin.
If for example IHP is used as allosteric effector, first, lipid vesicles are loaded with IHP and then fused with erythrocytes after which IHP is bound to the haemo-globin whereby the allosteric conformation of the haemo-globin and therewith its affinity to oxygen is changed.
The incorporation of the combination of allo-steric effectors and lipid vesicles into the erythrocytes is performed extracorporal.
During the application and use erythrocytes are separated from drawn blood, modified by the incorporation of lipid vesicles together with allosteric effectors and the modified erythrocytes refed to the blood plasma. There-fore, it is possible to preserve stored blood containing modified erythrocytes.
The modified erythrocytes can be used also included in a physiologic carrier for injection or trans-fusion into a blood circle.

... .

113~7Z4 A specific manner to prepare modified erythro-cytes according to the invention can be described as follows:
a) Inositol hexaphosphate is dissolved in an iso-tonic buffer until the solution is saturated, b) the mixture of lipids is suspended in the solu-tion of a), the mole ratio of phosphatidylcholine, phosphatidylserine and cholesterol being in the range of 10 to 5 : 4 to 1 : 10 to 3, c) the sole prepared suspension is disintegrated by ultrasonic treatment or by an injection process, d) The mixture is centrifuged and the upper sus-pension separated. This upper suspension con-tains the inositol hexaphosphate rich, small lipid vesicles as well as free inositol hexa-phosphate, e) Human erythrocytes separated from blood plasma by centrifuging are suspended in the upper sus-pension of d), f) during incubation the vesicles fuse in~o the erythrocytes, g) the now modified intact erythrocytes are washed with isotonic NaCl solution or isotonic buffer, whereby free inositoi hexaphosphate are quanti-tatively removed. Thereafter the prepared eryth-rocytes are suspended in blood plasma or blood substitute.

113~7Z~

Following is a brief description of the drawings:
Fig. 1: Increase of 2 affinity of intra-erythrocytic haemoglobin with storage time at 4C. The 2 half-saturation pressure, PO (1/2) was measured at 25C in the absence of CO2.
Red blood cells (RBC) stored in ACD, ~--and in isotonic 0.10 M bis-Tris buffer pH 7.4 containing 0.154 molar NaCl c + _~_ respectively.
Fig. 2: "Left-shift" of the O2-binding curves of intra-erythrocytic haemoglobin depending on the storage time at 4C. RBC stored in isotonic 0.10 M bis-Tris buffer pH 7.4 containing 0.154 molar NaCl at 4C. Binding isotherms were measured at 25C
in the absence of CO2.
RBC totally depleted of polyphosphates;-------:
RBC at half-life time of polyphosphate depletion (~ 1/2 = 9 d); -.-.-.-.-: fresh RBC. Arrow indi-cates desaturation of haemoglobin under the 2-partial pressure of 30 mm Hg.
Fig. 3: p~ dependence of O2-binding curves at ~5C in the absence of CO2. RBC stored 17 days at 4C
in isotonic 0.10 M bis-Tris buffer pH 7.4 con-taining 0.154 molar NaCl: --~ --, pH 7.72;
-: pH 7.42; -.-.-.-.-.: pH 7.08. Arrow indicates desaturation of haemoglobin under the O2-partial pressure of 30 mm Hg.
Fig. 4: Bohr effect of intra-erythrocytic haemoglobin at 25C in the absence of C02. RBC stored 17 days at 4C in isotonic 0.10 }nolar bis-Tris buffer pH 7.4 containing 0.154 molar NaCl.
. g 113~724 Fig. 5: Irreversible IHP incorporation into red cells;
2 half-saturation pressure of erythrocytes measured at 25C and in the absence of CO2 be-fore and after V2-mediated IHP incorporation at ` pH 7 . 6 . RBC stored at 4C in isotonic 0.10 bis-Tris buffer pH 7.4 containing 0.154 molar NaCl;
~ : polyphosphate depletion curve of RBC; ~3~ IHP incorporation at pH 7.6 and storage of IHP-loaded RBC at 4C; ~
IHP incorporation at pH 7. 6 and storage of IHP-loaded RBC at 37C. IHP incorporation carried out under standard conditions.
Fig. 6: Bohr effect of IHP-loaded erythrocytes at 25C
in the absence of CO2. ~C-~c-~c- :
V2-mediated IHP incorporation at pH 7.6;
~ -: V2-mediated IHP incorporation at pH 7.8. RBC stored at 4C in isotonic 0.10 molar bis-Tris buffer pH 7.4 containing 0.154 molar NaC1; IHP incorporation carried out under stan-dard conditions.
~ig. 7: Influence of vesicle composition on the "right-shift" o~ O2-binding isotherms measured for pH
7.6 at 25C in the absence of CO2. IHP
incorporation at pH 7.6 was carried out under standard conditions and cells were suspended in isotonic buffer pH 7.6.
-.-. - .-.- .-: RBC 19 days stored in isotonic 0.10 molar bis-Tris buffer pH 7.4 containing 0.154 molar NaCl; ----------: RBC after Vl-mediated IHP incorporation; ^- - : fresh RBC;

~130724 ~ RBC after V2-mediated IHP incor-poration. Arrow indicates desaturation of haemo-globin under the O2-partial pressure of 30 mm Hg.
Fig. 8: Stability of IHP-loaded vesicles at 37C.
~ ~ ~ V2 vesicle; D ~ n V3 vesicle. Vesicle-mediated IHP uptake by RBC
at pH 7.4 under standard conditions. PO (1/2) of IHP-loaded erythrocytes measured at 25C in the absence of CO2.
Fig. 9: Kinetics of incorporation of 14C-cholesterol from vesicles into intact erythrocytes. Incubation was carried out at 37C in isotonic 0.1 molar bis-Tris buffer pH 7.4 containing 0.154 molar NaCl.
Fig. 10: Kinetics of incorporation of 14C-cholesterol from V2-vesicles into intact erythrocytes. Radio-activity measured in Folch extracted lipids of the erythrocytes. Conditions of incubation as in Fig. 9.
Fig. 11: Kinetics o~ 14C-cnolesterol uptake by Hela cells from lipid vesicles. Incubation at 37C in phos-phate buffered saline pH = 7.4.
Fig. 12: pH dependence of 14C-cholesterol uptake by Hela cells from vesicles. Conditions as in Fig. 11.
Fig. 13: Influence of pH of incubation medium on the 2-half-saturation pressure of IHP-loaded erythro-cates. PO (1/2) measured at 25C in the absence of CO2.
Fig. 14: Kinetics of the V2-mediated IHP incorporation by erythrocytes at pH 7.38 in the presence of IHP in the outer medium. Incubation of 57 days 113~7Z4 old RBC was carried out under standard condi-tions. PO (1/2) was measured at 25C in the absence of CO2.
Fig. 15: Influence of pH of incubation medium on the half-life time of V2-media~ed IHP incorporation.
RBC were incubated under standard conditions with IHP in the outer medium. PO (1/2) measuxed at 25C in the absence of CO2.
Fig. 16: Kinetics of the V2-mediated IHP incorporation by erythrocytes at pH 7.4 in the absence of IHP
in the outer medium. Incubation was carried out under standard conditions. PO (1/2) was measured at 25C in the absence of CO2.
Fig. 17: O2-binding curves before and after V2-mediated IHP incorporation by erythrocytes at pH 7.4.
Incubation was carried out under standard conditions at pH 7.6. O2-release by the erythro-cytes against the brain critical O2-partial pressure of 30 mm Hg is indicated by arrows:
At 25C: , erythrocytes, 41 days old, (5% 2 release);-------, IHP-loaded erythrocytes (47% 2 release); at 37C -.-.-.-, erythrocytes, 41 days old, (21~ 2 release); -...-... .
IHP-loaded erythrocytes (80% O2release).
The degree of oxygen desaturation of intra-erythro-cytic haemoglobin in the capillaries depends not only on the venous O2-partial pressure but above all on the oxygen affinity of the haemoglobin in the red cells. Intra-erythro-cytic allosteric effectors controlling the 2 affinity are Bohr protons, CO2 and organic phosphate compounds, 113(~7~4 particularly DPG (2,3-bisphosphoglycerate-natural allosteric effec~or) and ATP (adenosine triphosphate).
The depletion of DPG and ATP in stored red cells leads to a progressi~7e increase of the oxygen affinity (Balcerzak, S. et al. (1972) Adv. Exp. Med. Biol. 28, 433 - 447) demonstrated in Fig. 1 by plotting the 2-partial pressure at half-saturation (measured at 25C) versus the storage time (erythrocytes stored at 4C). The O2-binding isotherms are measured in the absence of CO2 and at constant pH (pH 7.4) in order to preclude influences of these allosteric effectors on the half-saturation pres-sure. The end point of the progressive polyphosphate de-pletion is defined by PO (1/2) = 4.2 mm Hg, which i5 the half-saturation pressure of totally phosphate-free (stripped) haemoglobin; the starting point, i.e. PO (1/2) of fresh erythrocytes, depends on the composition of the suspending medium. From these polyphosphate depletion curves a new functional parameter of stored erythrocytes can be deter-mined, the so-called half-life timc of intra-erythrocytic polyphosphate. It is 9 d (days) in isotonic 0.1 M bis-Tris buffer pH 7.4 and 12 d (days) in ~CD (acid-citrate-dextrose: conservation solution) stabilisator solution.
The depletion of polyphosphates in erythrocytes causes a "left-shift" of the O2-binding isotherm and there-fore a decrease of the capacity of oxygen release.
The "left-shift" of the O2-binding curve and the decrease of the capacity of oxygen release of at 4C stored er~7throcytes at 25C are shown in Fig. 2. Under an 2-partial pressure of 30 mm Hg the desaturation of fresh ~,;

113~ 4 erythrocytes reaches 11~, whereas erythrocytes being half-depleted of polyphosphates desaturate only to 1%.
The "left-shift" of the O2-binding curves of poly-phosphate-depleted erythrocytes causes impairing of oxygen delivery In the tissu~s. Thus a massive transfusion of stored blood (being DPG deficient) results in a fall in muscle pH and an increase of the lactate level in the plasma accompanied by a fall of the blood pressure known as the "Transfusion-Syndrome" (Kevy, S.V. et al. (1972) Adv. Exp.
Med. Biol. 28, 511 - 516). This decrease in pH only par-tially counter-regulates the "left-shift" demonstrated in Fig. 3. For an 02-partial pressure of 30 mm Hg shift of the pH from 7.42 to 7.08 increases the oxygen desaturation of red cells (stored for 17 d at 4C in isotonic 0.1 M bis-Tris buffer pH 7.4) from 1~ to 5%; but a desaturation to 11% observed with fresh red cells at pH 7.46 cannot be effected by the Bohr effect alone (see Fig. 2). The influ-ence of pH on the 2 affinity (Bohr effect) oE stored red cells suspended in isotonic 0.1 M bis-Tris and Tris buffers, respectively, is shown in Fig. 4 for the range of p~ 7.0 to 7.8. The 02-binding curves are measured at 25C and in the absence of CO2 with erythrocytes stored for 17 days at 4C. These aged erythrocytes have lost more than half of their poly-phosphate effect on the 2 affinity of haemo-globin (see Fig. 1). The Bohr effect,-~ Po (1/2)/a pH, amounts to 0.53 protons per mole 2 The number of Bohr protons released with oxygen binding is constant at least up to an RBC age of 34 days when already 75~ of the poly-phosphate effect is lost. Therefore storage, i.e. depletion of polyphosphates, has no effect on the Bohr effect of the erythrocytes.

~13~'724 Methods Collection and storage of human blood:
A volume of 100 ml blood was drawn from a young healthy volunteer and collected into a 250 ml-Bioflask (Biotest-Serum-Institut, Frankfurt a. Main) containing 50 ml ACD stabilisator. This sample was stored at 4C.
Collection and storage of human erythrocytes:
A volume of 300 ml blood drawn from a young pro-band was collected into a plastic bag which contained heparin or sodium citrate for preventing blood clotting.
The blood sample was chilled in an ice bath and further work was carried out at 4C. Erythrocytes were separated from the plasma by centrifugation at 23500xg for 20 minutes (Sorvall, Type RC-2Bi Rotor SS 34; 12000 rpm). The packed red cells were suspended in isotonic pH 7.4 saline bis-Tris buffer (0.10 molar bis-Tris, 0.154 molar NaCl) and centrifuged; this washing procedure was repeated three times. Finally, the packed erythrocytes were suspended in 300 ml isotonic saline bis-Tris buffer pH 7.4 and stored at 4C.
pH measurement and adjustment of the erythrocyte susPension:
The red cells stored in isotonic 0.1 M buffers pH
7.4 and ACD respectively were centrifuged at room tempera-ture for 2 minutes at 8000xg (Eppendorf Centrifuge, Type 3200; 12000 rpm). The desired pH of the erythrocyte sus-pension was adjusted by repeated exchange of buffering medium: The cells were suspended in the desired buffer and ag2in centrifuged; this procedure has to be repeated until a constant pH is reached.

1~3g~724 The pH is measured at 25C with a glass electrode (ingold, Frankfurt a. Main, Typ. 406-M3, a = 35 mm). The accuracy of the pH measurement is + 0.02 units.
Preparation of the IHP-loaded lipid vesicles:
IHP was dissolved for instance between room tem-perature and 50C in an isotonic bis-Tris bufIer (0.10 molar bis-Tris, 0.154 molar NaCl) pH = 7.4 up to saturation (0.19 M). A lipid mixture consisting of phosphatidylcholine (PC) : phosphatidylserine (PS) : cholesterol (Ch) in the molar ratios 8 : 2 : 7 (see Table 1) was suspended in this solution and sonicated 45 min under nitrogen at ~50C. The temperature range for vesicle preparation is limited only by the freezing point of the buffer and by the thermal stability of the polyphosphate. The sonication ~as per-formed with a ultrasonic disintegrator (Scholler, Type 125, Frankfurt a. Main) with a titan dip-probe (10 kHz). Soni-cation can be effectively performed at energies preferably above 100 W/cm2. After sonication the vesicle suspension ~as centrifuged for 1 h at 100000xg at 25C in an ultracen-trifuge (Beckmann, Typ L5-65, Rotor 60). The supernatant contains the small lipid vesicles, with a diameter of 500 A. When the vesicles are formed they include the solution in which the lipids are suspended.
Table 1: Composition of the lipid vesicles 25~esicles PC : PS : Ch molar ratios .
Vl 9 : 1 : 8 V2 8 : 2 : 7 V3 8 : 4 : 7 V4 8 : 0 : 7 Operable molar ratios ranges (Vl - V3) = PC : PS : Ch =

10-5 : ~-1 : 10-3.

Phosphatidylserine was from bovine brain (Koch-Light, GB) and phosphatidylcholine from egg yolk (Lipid Special~ies, Boston, U.S.A.). Cholesterol and the sodium salt of inositol hexaphosphate were purchased from Merck (Dar~.stadt) and Sigma (Munchen) respectively. All lipids were puri~ied by column chromatography and their purity was checked by thin layer chromatography. 1 nmol/l of lipid yields 2 x 1011 lipid bilayer vesicles (small vesicles, d ~ 500 A). 2 x 1011 lipid vesicles were incubated with 106 erythrocytes, but other ratios were used as well, as it will be seen below.
Incorporation of inositol hexaphosphate into human erythrocytes:
For in vitro experiments a volume of 200 ~1 eryth-rocy~e suspension was centrifuged at room temperature for 2 min at 8000xg (Eppendorf Centrifuge, Type 3200, 12000 rpm).
If necessary, the packed cells were washed and adjusted to the desired pH as described above and then resuspended in a volu~e of 200 ,ul isotonic 0.1 M buffer of desired pH. To this an equal volume of the lipid vesicle suspension of desired ?H ~as added. The erythrocytes were incubated for 1 h at 37C. The ~rythrocytes were then repeatedly washed with isotonic 0.1 M bufer until a constant pH value was reached. Precipitation tests with Ca were carried out with the supernatant until no free IHP could be detected anymore.
Any ~u fer system effective in pH range 7 to 8 which does not af ect the structural, morphological and functional intesrity of the erythrocytes may be used.

~1307Z4 Measurement of the O2-binding curves:

O2-binding curves were measured at 25C by means of the rapid diffusion technique (Sick, H. and Gersonde, K.

(1969), Analyt. Biochem. 32, 362 - 376; Sick, H. and Gersonde, K. (1972), 47, 46 - 56).
Results Bohr effect of stored human red cells after fusion with vesicles-.
2 half-saturation pressure, cooperatively (n = 2.8) and Bohr effect of human erythrocytes stored for 40 days in isotonic buffering medium pH 7.4 at 4C and par-tially depleted of polyphosphates are not significantly changed after incubation in isotonic pH 7.6 buffer with vesicles V2 at 37C for 1 h. Furthermore, incubation of erythrocytes with vesicles having different lipid composi-tions (Vl and V3) has also no effect on the O2-binding par-ameters of the intra-erythrocytic haemoglobin.

Bohr effect of stored human red cells after vesicle-mediated (V2) incorporation of ino-sitol hexaphos~hate:
IHP, the strongest allosteric effector of haemo-globin ~nown up to llOW decreases the 2 affinity of haemo-globin indicated by a "right'shift" of the O2-binding curve.
Human erythrocytes not being able to synthesize IHP can be loaded with this polyphosphate during fusion with lipid vesicles containing the effector. The experiments described by this invention exemplified by IHP incorporation would essentially go in the same direction with all kinds of allo-steric effectors, f.i. sugar phosphates as inositol penta-phosphate, inositol tetraphosphate, inositol triphosphate, inositol diphosphate, and diphosphatidylinositol diphos-phate, further polyphosphates as nucleotide tri-, di-, and 113~72~

monophosphates, ~lcohol phosphate esters. Inorganic anions (hexacyanoferrat(II)phosphate and chloride) with effector properties, as well as mixtures thereof, which are unable to cross the erythrocyte membrane can be used. Also in specific cases of haemoglobin which show a mutation, organic anions as polycarboxylic acids can be used as allosteric effectors. As example for the polycarboxylic acid maleic acid can be used in cases of "Zurich" haemoglobin.
Human erythrocytes stored at 4C in isotonic buf-fering medium pH 7.4 for 25 days (PO (1/2) = 6.0 mm Hg~
were adjusted to pH 7.6 (PO (1/2) = 4.5 mm Hg) and then incubated for 1 h at 37C in isotonic pH 7.6 0.1 molar Tris buffer containing 0.19 molar IHP wlth IHP-loaded V2 vesicles.
After IHP incorporation into erythrocytes the 2 half-satu-ration pressure increases drastically (PO (1/2) = 14.3 mm Hg) by a factor of 3.2 and exceeds the value for fresh erythro-cytes. (PO (1/2) = 10.55 mm Hg) by a factor of 1.4. Then these IHP-loaded erythrocytes were stored at 4C in isotonic pH 7.6 buffering medium for 6 days; the half-saturation pressure remained constant. After further 4 days storage at 4C the IHP-loaded erythrocytes were changed to pH 7.28 and the PO (1/2) increased again to 32.1 mm Hg. In other experiments, washed erythrocytes stored at 4C for 36 days (more depleted of DPG) were adjusted to pH 7.6 and IHP
incorporated as described above. Again the PO (1/2) in-creased to 14.0 mm Hg. The IHP-loaded cells were stored at 37C for 2 days. No change in 2 affinity was observed either after storing these IHP-loaded cells at 37C for 2 days. Contrary to normal erythrocytes which show during storage at 4C half-depletion of physiological polyphos-phates, IHP-loaded erythrocytes do not seem to hydrolyse , 113~724 IHP during at least 9 days indicated by a constant PO (1/2) value (see Fig. 5). Thus, substantially longer storage times can be achieved with the so treated erythrocytes.
In addition to the IHP-loaded red cells are show-ing a Bohr effect larger than of the non-treated cells (see Fig. 6).
In the absence of CO2 the Bohr effect of erythro-cytes incubated with IHP-loaded V2 vesicles in the presence of ~ree IHP at pH 7.6 and adjusted to the desired pH by washing with the respective isotonic 0.1 M buffer amounts to -a Po (1/2)/~pH = 1.20 protons per mole 2 The Bohr proton release of IHP-loaded erythrocytes is 3 times larger than in normal fresh red cells. Therefore IHP-loaded cells make the oxygen release in the tissues and the oxygen up-take in the lungs more efficient. Incorporation of IHPwith IHP-loaded V2 vesicles in the presence of free IHP is much more efficient at pH 7.8. After having changed the pH of the IHP-loaded cells to 7.4 the theoretically expected increase to P~ (1/2) = 35 mm Hg can be observed (see Fig. 6).
On tne other hand the Bohr effect seems to become smaller ~ Po (l/2)/a p~l - 0.9 protons per mole 2) after incor-por2tion at pH 7.8.

Effect of vesicle composition on the inositol hexaphosphate uptake by human erythrocytes: _ The amount of IHP taken up by human erythrocytes is m~asured as the extent of "right-shift" of the 2-binding curve. Thus the 2 half-saturation pressure measured under standard conditions is an expression of the efficiency of i~corporation. This efficiency of the IHP uptake depends lars~ly on the lipid composition of the vesicles.

1~3CJ 724 In Fig. 7 the "right-shift" of the O2-binding curve is demonstrated for the Vl and V2 vesicles after incubation at pH 7.6. V2 vesicles show the largest "righ~-shift" with a desaturation o~ 14% at 30 rnm Hg. Erythro-cytes, 19 days old, show no desaturation at this 2 pres-sure, fresh red cells, however, desaturate to 5~. The incorporation of IHP with V2 vesicles improves the 2 delivery of the normal red cells at 30 mm Hg by a factor of about 3. V2 and V3 vesicles induce identical IHP uptake by erythrocytes and therefore identical "right-shifts" of the O2-binding curves.
In the absence of IHP in the outer medium Vl, V2 and V3 vesicles, having included IHP, do not differ with regard to their half-life time of incorporation, ~-1/2' being 30 min. Vl and V3 show only less than the half of the IHP effect measured for dialyzed IHP-loaded V2 vesicles.
This indicates a reduced stability of Vl and V3.
In Fig. 8 the difference in stability of V2 and V3 is demonstrated~ V2 has a half-life time of sta~ility of about 3 days, V3 of about 1.5 days.
Incor~oration o~ the vesicles into cells:
The incorporation of the lipid vesicles into intact erythrocytes was followed with the vesicles Vl, V2, V3 labelled with 14C-cholesterol or 14C-phosphatidylcholine.
2; This was compared with the incorporation of the same vesi-cles in cultured Hela cells. The radioactivity was assayed both in the intact erythrocytes (by solubilization and bleaching with a Lumac reagents kit) and in their total lipid Folch extracts. The incorporation was followed over a pe~iod of 4 hours. The results are shown in Fig. 9 and 113~724 10. Fig. 9 shows the data for the vesicles Vl, V2 and V3 with intact erythrocytes. The incubation medlum con-tained 10 ml RBC (red blood cells), 10 ml IHP-loaded vesi-cles in isotonic 0.1 M bis-Tris buffer pH 7.4. Aliquots were taken after 10, 20, 40, 60, 90, 120, 180, 240 minutes and counted. The half-life time of incorporation is 45 min for the V2 vesicles (which show also the highest radio-activity level in the RBC) and 35 min for Vl and V3. It has to be stressed that the radioactivity found in the RBC
does not necessarily indicate vesicle incorporation, as it is well known (Bloj, B. and Zilversmit, D. (1977) Biochem-istry 16, 3943 - 3948) that cholesterol exchanges between vesicles and erythrocytes. In the RBC lipid extracts (see Fig. 10) we find a ~1/2 of 30 min when V2 vesicles were used.
~en V2 and V3 vesicles are incorporated into Hela cells the same pattern is obtained (see Fig. 11). The incubation of the Hela cells with the vesicles was made under the same comditions as described before. In another set of experi-ments, Hela cells were incubated with 4C-cholesterol-con-taining vesicles in isotonic buffers, at several pH values between 7 and 8. Fig. 12 shows that the pH variations between 7 - 8 had little influence on the incorporation of the labelled lipid by the cells.
The half-life time of the radioactivity uptake by the erythrocytes, when incubated with radioactively labelled vesicles is the same as the half-life time of the IHP uptake by erythrocytes incubated with dialyzed, IHP-loaded vesicles (Fig. 16). This is additional proof that we measure not only lipid exchange between cells and vesicles but fusion of vesicles with the cells.

~3~724 Thin layer chromatograms of the erythrocyte li?id extract showed the enrichment of the R~C membrane li?ids with the lipids of the vesicles.
Apparent pH-optimum of the V2-mediated IHP
uptake by strored human red cells._ The uptake of IHP by washed erythrocytes depends on the pH of the incubation medium. Plotting PO (1/2) of IHP-loaded erythrocytes versus pH of incubation medium (see Fig. 13) demonstrates an apparent pH optimum of IHP incor-poration in the range of pH 7.4 to 7.5. The decrease of PO (1/2) above pH 7.5 corresponds to the Bohr effect curve shown in Fig. 6 and correlates with the decrease of IHP
affinity to haemoglobin. Below pH 7.4 the dramatic decrease of P0 (1/2) indicates a limited IHP incorporation and there-fore the theoretically expected PO (1/2) for totally trans-formed haemoglobin by bound IHP is not observed.
The change of buffering medium to pH 7.4 after incubation at pH 7.8 increases the PO (1/2) of intra-erythrocytic haemoglobin to values of 30 to 40 mm Hg at 25C
as demonstrated in Fig. 6. Incubation at pH 7.8 and buffer-ing to different pH-values lead to an increase of PO (1/2) over the whole range of pH. From this result we can con-clude that incorporation of IHP into erythrocytes is more ef ective above pH 7.4 although at lower pH values are also efective.
Kinetics of the IHP uptake by stored human red cells:
Fig. 14 shows the time-dependent decrease of the 2 affinity of erythrocytes after incubation with IHP-loaded V2 vesicles in 0.19 M IHP solution at pH 7.35. The increase ~13~72~L

of PO (1/2) reaches its half-maximum value after 4 min. The kinetics of IHP incorporation measured as increase of PO (l/2) depends on the pH of the incubation medium. In Fig. 15 the half-life time of IHP incorporation is plotted versus pH. The uptake of IHP is a slower reaction at lo-`~7 pH (pH 7.3) and a faster reaction at higher pH (pH 7.7).
The short half-life time of incorporation at high pH corres-ponds to the larger amount of IHP incorporated into erythro-cytes (c.f. Figures 6 and 13).
The kinetics of IHP incorporation is strongly influenced by the presence of free IHP in the outer medium.
Removal of free IHP by dialysis or gel filtration of the vesicle suspension leads to an increase of the half-life ti~e of IHP incorporation to 30 min at pH 7.4 (see Fig. 16).
ATP level in IHP-loaded erythrocytes:
The adenosine triphosphate (ATP) content of erythrocytes is of ~reat interest from the viewpoint of red cell preservation and of intact function. The ATP
level was measured in red cells havin~ incorporated empty V2 vesicles and IHP-loaded V2 vesicles under the conditions described. All measurements were carried out in isotonic 0.1 M bis-Tris buffer pH 7.4. The ATP concentration was measured with the luciferin-luci~erase system:

Enzyme + luciferin + ATP ~____ E-S-AMP + pyrophosphate (E) (S) __ ___l E + oxidized substrate + A~lP + C02 + h ~
The reaction is so efficient that one proton is produced for each ATP molecule utilized. The incorporation o~
empty vesicles or of IHP-loaded vesicles is without signif-icant influence on the ATP level in the erythrocytes.

`,;~i ~, , .

Table 2: ATP content of erythrocytes.
r ATP
l,uM/ml RBC~
_ Erythrocytes 0.92 + 10%
Erythrocytes-V2 0.84 Erythrocytes-V2-IHP 0.93 . . _ . .

The data are for one week-old RBC. The lack of change o-^
the ATP level in the RBC after incorporation of IHP indi-cates unaltered cell-viability, functionality and plas-ticity of the RBC.
02-releasing effect by fusion of IHP-loaded veslcles with erythrocytes:
The "right-shift" of the O2-binding curves after incorporation of IHP is shown in Fig. 17. After fusion of 41-day old erythrocytes, suspended in isotonic bis-Tris buffer, pH = 7.4, with the IHP-loaded V2 vesicles the 2 half-saturation pressure increases from 7 to 28 ~m Hg.
This means that the normal but aged erythrocytes are loaded at 2;C to 95~ with 2 under an O2-partial pressure of 30 ~ Hg, whereas the IHP-loaded erythrocytes contain only 53% of oxygenated haemoglobin under the same conditions.
About 60% of the haemoglobin in the erythrocytes has bourd IHP after fusion of the IHP-loaded vesicles with the erythrocytes.
Under physiological conditions (at 37C) a 2 half-saturation pressure of 60 mm Hg is computed for the IHP-loaded erythrocytes at pH 7.4. Under a critical 2-partial pressure of 30 mm Hg in the brain, 80% of the hae-moglobin from vesicle-treated erythrocytes would release -the bound 2~ while normal, untreated erythrocytes would release ~1 3~724 under these conditions only 20 to 25% of the oxygen. The effective affinity of the erythrocytes can be varied bet~7een these two limits either by varying the IHP concentra,ion in the lipid vesicles or the ratio of treated to untreated erythrocytes in bloo~.
This result shows that the method which we pro-pose in order to incorporate IHP into the erythrocytes pro-vides a lasting, significant and controlled lowering of the 2 affinity of haemoglobin in intact cells. The erythro-cytes thus IHP-loaded are particularly suitable for the cortrol of the 2 supply of the tissues in the cases men-tioned above.
Hi~h-altitude adaptation of rats and dogs:
A 200 g rat (body weight) with a blood volume of 14 ml (PO (1/2) = 14.0 mm Hg at 25C and pH 7.4) was kept in a chamber under decreasing O2-partial pressure. At an 02-partial pressure of 120 mm Hg equivalent to an altitude of 13200 m the rat tumbled down because of 2 deficiency in the muscles of extremities. Then tne 2 pressure in the cha~rber was quickly restored to the normal value and the rat behaved normally. From this animal 1 ml blood was collected, the erythrocytes were isolated and loaded with IHP as described in Methods. The IHP-loaded erythrocytes were resuspended in the plasma (PO (1/2) = 28.0 ,~m Hg at 25C and pH 7.4) and then retransfused to the rat. After having now decreased the O2-partial pressure the rat tumbled down at 100 mm Hg (- 14200 m altitude). This treat-ment therefore caused an increase of the altitude ceilins of *8%. The altitude adaptation experiment was repeated with this rat 24 h later and lead to the same result.

~L~3~724 In another experiment a 9 kg (body weight) dog with a blood volume of 630 ml (PO (1/2) = 10.8 mm Hg at 25C and pH 7.4) was kept in a chamber under decreasing O2-partial pressure. At an O2-partial pressure of 140 mm Hg (-~ 12200 m altitude) the dog tumbled down. Then the 2 pressure in the chamber was quickly restored to the normal value and the dog behaved normally. From this do~ 100 ml blood were collected, the erythrocytes were isolated and loa~ed with IHP as described in Methods. The IHP-loaded erythrocytes were resuspended in the serum (PO (1/2) =
15.0 mm Hg at 25C and pH 7.4) and then retransfused to the dog. After having decreased the O2-partial pressure the dog tumbled down at 110 mm Hg (~- 13800 m altitude).
Therefore this treatment caused an increase of the alti-tude ceiling of ~-13%. This altitude ceiling was measured over 2 days with the same result.
Both animals were alive and well when last observed, four months (rat) and one month (dog) after the experiments.

Human erythrocytes with high affinity mutant haemo~lobin:

A 19 year old female patient with a haemoglobin mutant of unknown structure (HbMainz) with high 2 affinity donated blood. At 25C pH 7.4 her fresh erythrocytes showed a PO (1/2) = 7.5 mm Hg. Thus the oxygen supply to the tissues is decreased to 50%. Because of oxygen deficiency in her tissues, this patient receives blood transfusions every six weeks. Loading of these erythro-cytes with IHP, as described in Methods, led to an increase 1130~Z~

of PO (1/2) = 18.7 mm Hg. This shows that the high 2 affinity of this patient's blood can be decreased by our method to values being above the normal value of fresh erythrocytes corresponding to an increase of the oxygen supply of`+23~ of a t3tal blood exchange.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for modifying intact erythrocytes having improved O2-release which comprises fusing lipid vesicles loaded with allosteric effectors into erythrocytes containing heamoglobin and binding the allosteric effectors to the heamoglobin of the erythrocytes, the lipid vesicles comprising mixtures of phospha-tidyl choline:phosphatidylserine:cholesterol in a mole ratio of 10 to 5 : 4 to 1 : 10 to 3.

2. A process for the provision of erythrocytes having improved O2-release which comprises (1) dissolving inositol hexaphosphate in an isotonic buffer solution until the solu-tion is saturated, (2) suspending in this solution, a lipid mixture containing phosphatidylcholine : phosphatidylserine :
cholesterol in the mole ration 10 to 5 : 4 to 1 : 10 to 3, (3) subjecting the resulting suspension to ultrasonic disinte-gration, then (4) centrifuging to separate the supernatant suspension which contains the small inositol hexaphosphate-enriched lipid vesicles, after which (5) human erythrocytes, previously separated from the blood plasma by centrifugation, are resuspended in said supernatant suspension which contains said small inositol hexaphosphate-enriched lipid vesicles and (6) incubating the resulting suspension to effect fusion of said vesicles with said erythrocytes, and (7) washing the now modified intact erythrocytes with isotonic NaCl-solution or isotonic buffer quantitatively removing thereby free inositol hexaphosphate being still outside the erythrocytes and suspending the modified erythrocytes in blood-plasma or blood-plasma substitute.

3. A process of claim 2 wherein the ultrasonic disinte-gration is effected at energies above 100 w/cm2.

4. A process of claims 1 to 3 wherein said lipid mixture contains phosphatidylcholine : phosphatidylserine : cholesterol in the mole ratio 8 : 2 : 7.

5. A process of claims 1 to 3 wherein said lipid mixture contains phosphatidylcholine : phosphatidylserine : cholesterol in the mole ratio 9 : 1 : 8.

6. A process of claims 1 to 3 wherein said lipid mixture contains phosphatidylcholine : phosphatidylserine : cholesterol in the mole ratio 8 : 4 : 7.

7. A process as claimed in claim 1 for modifying intact erythrocytes having improved O2-release which comprises fusing thereto allosteric effector containing lipid vesicles in which the lipid consists essentially of phosphatidylcholine :
phosphatidylserine : cholesterol in the mole ratio 10 to 5 : 4 to 1 : 10 to 3.

8. Modified intact erythrocytes, whenever obtained according to a process as claimed in claim 7 or by an obvious chemical equivalent thereof.

9. Modified intact erythrocytes, whenever obtained according to a process as claimed in claim 1 or by an obvious chemical equivalent thereof.

10. A process as claimed in claim 1 for modifying intact erythrocytes having improved O2-release which comprises fusing thereto inositol hexaphosphate containg lipid vesicles in which the lipid consists essentially of phosphatidylcholine phosphatidylserine :cholesterol in the mole ratio of 8 : 2 : 7

11. Modified intact erythrocytes, whenever obtained according to a process as claimed in claim 10 or by an obvious chemical equivalent thereof.

12. A process as claimed in claim 1 for modifying intact erythrocytes having improved O2-release which comprises fusing thereto small inositol containing lipid vesicles in which the lipid consists essentially of phosphatidylcholine : phospha-tidylserine :cholesterol in the mole ratio 9 : 1 : 8.

13. Modified intact erythrocytes, whenever obtained according to a process as claimed in claim 12 or by an obvious chemical equivalent thereof.

14. A process as claimed in claim 1 for modifying intact erythrocytes having improved O2-release which comprises fusing thereto small inositol containing lipid vesicles in which the lipid consists essentially of phosphatidylcholine :
phosphatidylserine :cholesterol in the mole ratio 8 : 4 : 7.

15. Modified intact erythrocytes, whenever obtained according to a process as claimed in claim 14 or by an obvious chemical equivalent thereof.

16. A process as claimed in claim 1 for modifying intact erythrocytes having improved O2-release which comprises fusing thereto allosteric effector containing lipid vesicles in which the lipid consists essentially of phosphatidylcholine :
phosphatidylserine : cholesterol in the mole ratio 10 to 5 :
4 to 1 : 10 to 3 and the allosteric effectors are sugar phosphates selected from the group of inositol pentaphosphate, inositol tetraphosphate, inositol triphosphate, inositol diphos-phate and diphosphatidyl inositol diphosphate.

17. Modified intact erythrocytes, whenever obtained according to a process as claimed in claim 16 or by an obvious chemical equivalent thereof.

18. A process as claimed in claim 1 for modifying intact erythrocytes having improved O2-release which comprises fusing thereto allosteric effector containing lipid vesicles in which the lipid consists essentially of phosphatidylcholine :
phosphatidylserine : cholesterol in the mole ratio of 10 to 5 :

4 to 1 : 10 to 3 and the allosteric effector is a polyphos-phate selected from the group of nucleotide triphosphate, nucleotide diphosphate, nucleotide monophosphate and alcohol phosphate esters.

19. Modified intact erythrocytes, whenever obtained according to a process as claimed in claim 18 or by an obvious chemical equivalent thereof.

20. A process as claimed in claim 1 for modifying intact erythrocytes having improved O2-release which comprises fusing thereto allosteric effector containing lipid vesicles in which the lipid consists essentially of phosphatidylcholine :
phosphatidylserine :cholesterol in the mole ratio of 10 to 5 :
4 to 1 : 10 to 3 and in which the allosteric effector is an organic anion.

21. Modified intact erythrocytes, whenever obtained according to a process as claimed in claim 20 or by an obvious chemical equivalent thereof.

22. A process as claimed in claim 1 for modifying intact erythrocytes having improved O2-release in which the allosteric effector is selected from the group of inorganic anions.

23. A modified intact erythrocyte, whenever obtained according to a process as claimed in claim 22 or by an obvious chemical equivalent thereof.