IE47329B1 - Controlled improvement of the o2-release by intact erythrocytes - Google Patents

Abstract

1. Modified intact erythrocytes ensuring improved oxygen release of the blood, characterized in that allosteric effectors are irreversibly incorporated into the erythrocytes by means of lipid vesicles and these allosteric effectors are bound to the haemoglobin in the erythrocytes.

Description

One of the major functions of erythrocytes consists 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 0, The Oj-partial pressure in the lung is ''-'IOO mm Hg, in the capillary system <~70 mg Hg against which 02 must be dissociated from the oxygenated haemoglobin. Under physiological conditions only about 25% of the oxygenated haemoglobin may be deoxygenated; 75% is carried back to the lungs with the venous blood. Thus, the major fraction of the Hb-02 adduct is not used for the 02 transport.

Interactions of Hb with allosteric effectors enable an adaptation to the physiological requirement of maximum 02 release from the Hb-02 adduct with simultaneous conservation of the highest possible 02-partial pressure in the capillary system.

Hb's allosteric properties are responsible for the sigmoid shape of the 02-binding curve. Its half-saturation pressure (ρθ (1/2)) is an indicator of the 02 affinity. The slope of the sigmoid curve in the range of 40 to 60% saturation presents the degree of cooperativity of the four 02- binding sites in Hb (Hill coefficient 20 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 02~partial pressure (so-called rightshift) would leave to a facilitated 02 release in the capillary system and to an improved oxygen supply to the 3 29 surrounding tissues. A right-shift is actually achieved in vivo by the enhancement of the 2,3-bisphosphoglycerate level in erythrocytes and its binding to haemoglobin. Binding of 2,3-bisphosphoglycerate to Hb decreases the 02 affinity and increases the 02 half-saturation pressure. 2,3-bis-phosphoglycerate synthesized in the glycolysis bypass in erythrocytes is an effector for slow adaptation to 02 deficiency. 2,3-bisphosphoglycerate increases the half-saturation pressure and 4.3 mm Hg (25°C) to p„ (1/2) = 23.7 mm Hg (37°C), and „ 2 12.0 Hg (25°C), respectively (Imai, K. and Yonetani, T. (1975), J. Biol. Chem. 250, 1093 - 1098). A significantly stronger decrease of the 02 affinity, i.e. enhancement of the 02 half-saturation 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 02 half-saturation pressure to ρθ (1/2) = 96.4 mm Hg (37°C), and ρθ (1/2) = 48.4 mm Hg (25°C), respectively. IHP, 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 ι membrane or by endocytosis (Papahadjopoulos, 0. et al. (1976) Nature 252, 163-165). The fusion plays an important part in a number 47339 of membrane processes like: Synaptic transmission, secretion, plasma membrane assembly, infection with enveloped viruses. The fusion of cells with lipid vesicles was achieved in cell cultures and with erythrocyte ghosts.

Treatment of ML cells (cell line-murine lymphoma) with vesicles containing IgG with a high neutralizing titer against Coxsackie virus A-2 L protected the cells against subsequent infection with this virus. 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 through, did not lead to unambiguous results since the lipid vesicles are incorporated indiscriminately by all cells or are quickly destroyed in the liver.

The use of lipid vesicles for the incorporation of allosteric effectors or of other substances into erythrocytes has not been reported so far. Many experiments have shown that lipid vesicles can be injected into animals without 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 IHP with haemoglobin 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 membrane, it was possible to transport allosteric effectors as IHP into erythrocytes where, due to its much higher binding constant it replaces 47328 2,3-bisphosphoglycerate at its binding site in haemoglobin. Under these conditions Hb in erythrocytes change into an allosteric conformation with a significantly lower 02 affinity.

The lowering of the 02 affinity, i.e. the enhancement of the 02 half-saturation pressure of haemoglobin in erythrocytes, increases the capacity of erythrocytes to dissociate the bound 02 even against higher 02-partial pressure and thus improves the 02 supply to the tissues.

Erythrocytes, having incorporated IHP and which therefore provide an improved 02 supply to the tissues may find their use in the following cases: 1. Under low Og-partial pressure in the inhaled air: Mountain climbers at high altitudes, astronautes in 02~poor atmosphere. 2. In the case of reduced Og-exchange surface: Decrease of the number of pulmonary alveoles in lung emphysema. 3. Increased resistance to 02 diffusion in the lung: Pneumonia, Asthma. 4. Decreased 02-transport capacity: Erythropeny, anaemic stages of all types, arteriovenous shunt.

. Blood circulation disturbances: Arteriosclerosis, thromboembolic processes, organ infarct, ischaemic states. 6. High 02 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 02-affinity of conserved blood: Transfusion, shock states. 9. Improvement of radiotherapy of cancer.

As allosteric effectors for the incorporation into erythrocytes are used which have a larger affinity to haemoglobin than the known physiologic effectors, 2,3-bisphosphoglycerate and adenosine triphosphate.

Specifically inositol hexaphosphate is preferred as allosteric effector.

Other sugar phosphates such as inositol’ pentaphosphate, inositol tetraphosphate, inositol triphosphate, inositol diphosphate and diphosphatidyl inositol diphosphate can be used as allosteric effectors.

Further allosteric effectors are other polyphosphates such as nucleotide triphosphates, nucleotide diphosphates, nucleotide monophosphates, and alcohol phosphate esters are suitable.

In case of certain mutations of haemoglobin, e.g. Zurich haemoglobin, organic anions as polycarboxylic acids can be used as allosteric effectors.

Finally, it is possible to use inorganic anions such as hexacyano ferrate, phosphate or chloride as allosteric effectors.

Lipid vesicles are mixtures of phosphatidycholine/ phosphatidylserine/cholesterol which are used in a mole ratio of to 5 : 4 to 1 : 10 to 3. Specifically favoured is a mole ratio, of 8:2 : 7, but also a mole ratio of 9 : 1 :8 7 329 as well as 8:4:7 is favourable.

As fluid carrier the customary liquid carrier, specifically buffered physiologic carriers are used which are known, e.g. 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 possible now that haemoglobin of the erythrocytes is transferred into an allosteric conformation which releases oxygen easier.

By the invention it is possible to produce modified 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 haemoglobin whereby the allosteric conformation of the haemoglobin and therewith its affinity to oxygen is changed.

The incorporation of the combination of allosteric effectors and lipid vesicles into the erythrocytes is conducted 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. Therefore, it is possible to preserve 3 39 stored blood containing modified erythrocytes.

The modified erythrocytes can be used also included in a physiologic carrier for injection or transfusion into a blood ci rcle.

A specific manner to prefer modified erythrocytes according to the invention can be described as follows: a) Inositol hexaphosphate is dissolved in an isotonic buffer until the solution is saturated, b) the mixture of lipid vesicles is suspended in the solution of a), the mole ratio of phosphatidylcholine, phosphatidylserine and cholesterol being in the range of 10 to 5: 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 suspension separated. This upper suspension contains the inositol hexaphosphate rich, small lipid vesicles as well as free inositol hexaphosphate, e) human erythrocytes separated from blood plasma by centrifuging are suspended in the upper suspension of d), f) during incubation the vesicles fuse into the erythrocytes, g) the now modified intact erythrocytes are washed with isotonic NaCl solution or isotonic buffer, whereby free inositol hexaphosphate are quantitatively, removed. Thereafter the prepared erythrocytes are suspended in blood plasma or blood substitute.

Following is a brief description of the drawings: Figure 1: Increase of 02 affinity of intra-erythrocytic haemoglobin with storage time at 4°C. The 02 half-saturation pressure, ρθ (1/2) was measured at 25°C in the absence of CO,,. Red blood cells (RBC) stored in ACD, —B—§3—β— and in isotonic 0.10 M bis-Tris buffer pH 7.4 containing 0.154 molar NaCl -o-— respectively.

Figure 2: Left-shift of the 02~binding curves of intraerythrocytic haemoglobin depending on the storage time at 4°C. RBC stored in isotonic 0.10 M bis-Tris buffer pH 7.4 containing 0.154 molar NaCl at 4°C. Binding isotherms were measured at 25°C in the absence of COg. - : RBC totally oepleted of polyphosphates;-------------; RBC at half-life time of polyphosphate depletion (χ = 9 d); ----------; fresh rbc. Arrow indicates desaturation of haemoglobin under the Og-partial pressure of 30 mm Hg.

Figure 3: pH dependence of 02-binding curves at 25°C in the absence of COg. RBC stored 17 days at 4°C in isotonic 0.10 m bis-Tris buffer pH 7.4 containing 0.154 molar NaCl:--------20 pH 7.72;-: pH 7.42;---------.: pH 7.08. Arrow indicates desaturation of haemoglobin under the Og-partial pressure of 30 mm Hg.

Figure 4 : Bohr effect of intra-erythrocytic haemoglobin at 25°C in the absence of COg. RBC stored 17 days at 4°C in 3.39 isotonic 0.10 molar bis-Tris buffer pH 7.4 containing 0.154 molar NaCl.

Figure 5: Irreversible IHP incorporation into red cells ; Og half-saturation pressure of erythrocytes measured at °C and in the absence of COg before and after V2-mediated IHP incorporation at pH 7.6. RBC stored at 4°C in isotonic 0.10 bis-Tris buffer pH 7.4 containing 0.154 molar NaCl; —0-0-0-: polyphosphate depletion curve of RBC; : IHP incorporation at pH 7.6 and storage of IHP-loaded RBC at 4°c: IH? incorporation at pH 7.6 and storage of IHP-loaded RBC at 37UC. IHP . incorporation carried out under standard conditions.

Figure 6 : Bohr effect of IHP-loaded erythrocytes at 25°C in the absence of COg. --0-0-0-: V2-mediated IHP incorporation at pH 7.6; -□—□—: V2-mediated IHP incorporation at pH 7.8. RBC stored at 4°C in isotonic 0.10 molar bis-Tris buffer pH 7.4 containing 0.154 molar NaCl; IHP incorporation carried out under standard conditions.

Figure 7 : Influence of vesicle composition on the rightshift of Og-binding isotherms measured for pH 7.6 at 25°C in the absence of COg. 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; - : RBC after V2-mediated IHP incorporation. 4733S Arrow indicates- desaturation of haemoglobin under the 02-partial pressure of 30 mm Hg.

V2 vesicle; -O-O-O—: V3 vesicle.

Vesicle-mediated IHP uptake by RBC at pH 7.4 under standard conditions. pn (1/2) u2 of IHP-loaded erythrocytes measured at 25°C in the absence of co2.

Figure 9 : Kinetics of incorporation of C-cholesterol from vesicles into intact erythrocytes. Incubation was carried out at 37°C in isotonic 0.1 molar bis-Tris buffer pH 7.4 containing 0.154 molar NaCl.

Figure 10 : Kinetics of incorporation of C-cholesterol from V2· vesicles into intact erythrocytes. Radio-activity measured in Folch extracted lipids of the erythrocytes. Conditions of incubation as in Figure 9.

Figure 11 : Kinetics of C-cholesterol uptake by He!a cells from lipid vesicles. Incubation at 37°C in phosphate buffered saline pH = 7.4.

Figure 12 ; pH dependence of C-cholesterol uptake by Hela cells from vesicles. Conditions as in Figure 11.

Figure 13 ; Influence of pH of incubation medium on the 02-half-saturation pressure of IHP-loaded erythrocytes, ρθ (1/2) measured at 25°C in the absence of C02. «1 Figure 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 old RBC was carried out under standard conditions. pn (1/2) was measured at 25°C in the u2 absence of COg.

Figure 15 : Influence of pH of incubation medium on the halflife time of V2-mediated IHP incorporation. RBC were incubated under standard conditions with IHP in the outer medium, ρθ (1/2) measured at 25°C in the absence of COg.

Figure 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, ρθ (1/2) was measured at 25°C in the absence of COg.

Figure 17 : Og-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. 02-release by the erytyrocytes against the brain critical Og-partial pressure of mm Hg is indicated by arrows: At 25°C:-, erythrocytes, days old, (5½ Og release); -------, IHP-loaded erythrocytes (47½ Og release); at 37°C--------, erythrocytes, 41 days old, (21% Og release);.-—-----, IHP-loaded erythrocytes (80% Og release). '4 7 329 The degree of oxygen desaturation of intra-erythrocytic haemoglobin in the capillaries depends not only on the venous Og-partial pressure but above all on the oxygen affinity of the haemoglobin in the red cells. Intra-erythrocytic allosteric effectors controlling the 02 affinity are Bohr protons, C02 and organic phosphate compounds particularly DPG (2,3-bisphosphoglycerate-natural allosteric effector) and ATP (adenosine triphosphate).

The depletion of DPG and ATP in stored red cells leads to a progressive increase of the oxygen affinity (Balcerzak, S. et al. (1972) Adv. Exp. Med. Biol. 28, 433-447) demonstrated in Figure 1 by plotting the 02-partial pressure at half-saturation (measured at 25°C) versus the storage time (erythrocytes stored at 4°C). The 02-binding isotherms are measuredin the absence of C02 and at constant pH (pH 7.4) in order to preclude influences of these allosteric effectors on the half-saturation pressure. The end point of the progressive polyphosphate depletion is defined by pn (1/2) = 4.2 mm Hg, which is the u2 half-saturation pressure of totally phosphate-free (stripped) haemoglobin; the starting point, i.e. ρθ (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 determined, the so-called half-life time 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 ACD (acid-citrate-dextrose : conversation solution) stabilisator solution. 339 The depletion of polyphosphates in erythrocytes causes a leftshift of the 02-binding isotherm and therefore a decrease of the capacity of °χ/9θη release.

The left-shift of the Og-binding curves and the decrease of 5 the capacity of oxygen release of stored-at-4°C erythrocytes at °C are shown in Figure 2. Under an Og-partial pressure of 30 mm Hg the desaturation of fresh erythrocytes reaches 11%, whereas erythrocytes being half-depleted of polyphosphates desaturate only to 1%.

The left-shift of the Og-binding curves of polyphosphatedepleted erythrocytes causes impairing of oxygen delivery in the tissues. 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. etal. (1972) Adv. Exp. Med. Biol. 28^511-516).

This decrease in pH only partially counter-regulates the left-shift demonstrated in Figure 3. For an Og-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 4°C 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 Figure 2). The influence of pH on the 02 affinity (Bohr effect) of stored red cells suspended in J7338 isotonic 0.1 M bis-Tris and Tris buffers, respectively, is shown in Figure 4 for the range of pH 7.0 to 7.8. The Og-binding curves are measured at 25°C and in the absence of C02 with erythrocytes stored for 17 days at 4°C. These aged erythrocytes have lost more than half of their polyphosphate effect on the 02 affinity of haemoglobin (see Figure 1). The Bohr effect, ~Δ Pn (1/2) /A pH, amounts to 0.53 protons per mole 0?. u2 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 polyphosphate effect is lost. Therefore storage, i.e. depletion of polyphosphates, has no effect on the Bohr effect of the erythrocytes.

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 4°C.

Collection and storage of human erythrocytes: A volume of 300 ml blood drawn from a young proband was collected 2V 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 4°C. Erythrocytes were separated from the plasma by centrifugation at 23500xg for 20 minutes (Sorvall, Type RC-2B; 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 4°C. pH measurement and adjustment of the erytyrocyte suspension: The red cells stored in isotonic 0.1 M buffers pH 7.4 and ACD respectively were centrifuged at room temperature for 2 minutes at 8000xg (Eppendorf Centrifuge, Type 3200; 12000 rpm). The desired pH of the erythrocyte suspension was adjusted by repeated exchange of buffering medium: The cells were suspended in the desired buffer and again centrifuged; this procedure has to be repeated until a constant pH is reached.

The pH is measured at 25°C with a glass electrode (Ingold, Frankfurt a. Main, Type 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 temperature and 50°C in an isotonic bis-Tris buffer (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) : phosphatidyl serine (PS) : cholesterol (Ch) in the molar ratios 8:2:7 (see Table 1) was suspended in this solution and sonicated 45 minutes under nitrogen at—1 50°C, 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 was performed with an ultrasonic disintegrator (Scholler, Type 125, Frankfort a. Main) with a titan dip-probe (10 kHz). 47339 Sonication can be effectively performed at energies preferably above 2 TOO W/cm . After sonication the vesicle suspension was centrifuged for 1 hour at 100000 xg at 25°C in an ultraeentrifuge (Beckmann, Type L5-65, Rotor 60). The supernatant contains the small lipid vesicles, with a diameter of < 500 8. When the vesicles are formed they include the solution in which the lipids are suspended.

Table I: Composition of the lipid vesicles + Vesicles PC : PS : Ch molar ratios VI 9 V2 8 V3 8 V4 8 +0perable molar ratios ranges (VI - V3) = PC : PS : Ch = 10-5 : 4-1 : 10-3.

Phosphatidylserine was from bovine brain (Koch-Light, GB) and phosphatidylcholine from egg yolk (Lipid Specialities, Boston, U.S.A.). Cholesterol and the sodium salt of inositol hexaphosphate were purchesed II from Merck (Darmstadt) and Sigma (Munchen) respectively. All lipids were purified by column chromatography and their purity was checked by thin layer chromatography. 1 nmol/1 of lipid yields 2 x 10^ lipid bilayer vesicles (small vesicles, di£ 500 A). 2 x 10^ lipid vesicles were incubated with 10° erythrocytes, but other ratios were used as well, as it will be seen below. 339 Incorporation of inositol hexaphosphate into human erythrocytes For in vitro experiments a volume of 200 μΐ erythrocyte suspension was centrifuged at room temperature for 2 minutes 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 volume of 200fil isotonic 0.1 M buffer of desired pH. To this an equal volume of the lipid vesicle suspension of desired pH was added.

The erythrocytes were incubated for 1 hour at 37°C. The erythrocytes were then repeatedly washed with isotonic 0.1 M buffer until a constant 2+ pH value was reached. Precipitation tests with Ca were carried out with the supernatant until no free IHP could be detected anymore. Any buffer system effective in pH range 7 to 8 which does not affect the structural, morpholical and functional integrity of the erythrocytes may be used.

Measurement of the 02~binding curves: 02-binding curves were measured at 25°C 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 02 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 4°C and partially depleted of polyphosphates are not significantly changed after incubation in isotonic pH 7.6 buffer with vesicles V2 at 37°C for 1 hour. Furthermore, incubation of erythrocytes with vesicles having different lipid compositions (VI and V3) 47333 has also no effect on the Og-binding parameters of the intraerythrocytic haemoglobin.

Bohr effect of stored human red cells after vesicle-mediated (V2) incorporation of inositol hexaphosphate IHP, the strongest allosteric effector of haemoglobin known up to now decreases the 02 affinity of haemoglobin indicated by a right-shift of the 02-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 allosteric effectors, e.g. sugar phospahtes as inositol pentaphosphate, inositol tetraphosphate, inositol triphosphate, inositol diphosphate, and di phosphatidyl inositol diphosphate, further polyphosphates as nucleotide tri-, di-, and monophosphates, alcohol phosphate esters. Inorganic anions (hexacyanoferrate (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 4°C in isotonic buffering medium pH 7.4 for 25 days (pn (1/2) = 6.0 mm Hg) were adjusted to pH 7.6 2 (pn (1/2) = 4.5 mm Hg) and then incubated for 1 hour at 37°C u2 - 47329 d in isotonic pH 7.6 0.1 molar Tris buffer containing 0.19 molar IHP with IHP-loaded V2 vesicles. After IHP incorporation into erythrocytes the 02 half-saturation pressure increases drastically (ρθ (1/2) = 14.3 mm Hg) by a factor of 3.2 and exceeds the value for fresh erythrocytes (pn (1/2) = 11.55 mm Hg) by a factor of 1.4. Then these 2 IHP-loaded erythrocytes were stored at 4°C in isotonic pH 7.6 buffering medium for 6 days; the half-saturation pressure remained constant.

After further 4 days storage at 4°C the IHP-loaded erythrocytes were changed to pH 7.28 and the pn (1/2) increased again to 32.1 mm Hg 2 In other experiments, washed erythrocytes stored at 4°C for 36 days (more depleted of DPG) were adjusted to pH 7.6 and IHP incorporated as described above. Again the pn (1/2) increased to 14.0 mm Hg. 2 The IHP-loaded cells were stored at 37°C for 2 days. No change in Og affinity was observed either after storing these IHP-loaded cells at 37°C for 2 days. Contrary to normal erythrocytes which show during storage at 4°C half-depletion of physiological polyphosphates, IHPloaded erythrocytes do not seem to hydrolyse IHP during at least 9 days indicated by a constant pn (1/2) value (see Figure 5). Thus, Ug substantially longer storage times can be achieved with the so treated erythrocytes.

In addition to the IHP-loaded red cells are showing a Bohr effect larger than of the non-treated cells (see Figure 6) .

In the absence of COg the Bohr effect of erythrocytes incubated with IHP-loaded V2 vesicles in the presence of free IHP at pH 7.6 and adjusted to the desired pH by washing with the respective isotonic 0.1 M buffer amounts of Δρθ (1/2)ApH = 1.20 protons per mole 0g. “47329 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 uptake in the lungs more efficient. Incorporation of IHP with 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 ρθ (1/2) = 35 mm Hg can be observed (see Figure 6). On the other hand the Bohr effect seems to become smaller (-Δ ρθ (1/2) /Δ,ρΗ = 0.9 protons per mole 02) after incorporation 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 measured as the extent of right-shift of the Og-binding curve. Thus the 02 halfsaturation pressure measured under standard conditions is an expression of the efficiency of incorporation. This efficiency of the IHP uptake depends largely on the lipid composition of the vesicles.

In Figure 7 the right-shift of the Og-binding curve is demonstrated for the VI and V2 vesicles after incubation at pH 7.6.

V2 vesicles show the largest right-shift with a desaturation of 14% at 30 mm Hg. Erythrocytes, 19 days old, show no desaturation at this 02 pressure, fresh red cells, however, desaturate to 5%. The incorporation of IHP with V2 vesicles improves the 02 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 Og-binding curves. 47333 In the absence of IHP in the outer medium VI, V2 and V3 vesicles, having included IHP, do not differ with regard to their half-life time of incorporation, -c^g, being 30 minutes. VI and V3 show only less than half of the IHP effect measured for dialyzed IHP-loaded V2 vesicles.

This indicates a reduced stability of VI and V3. In Figure 8 the difference in stability of V2 and V3 is demonstrated. V2 has a half-life time of stability of about 3 days, V3 of about 1.5 days.

Incorporation of the vesicles into cells: The incorporation of the lipid vesicles into intact erythrocytes was followed with the vesicles VI, V2, V3 labelled with 14 14 C-cholesterol or C-phosphatidylcholine. This was compared with the incorporation of the same vesicles in cultured Hela cells.

The radioactivity as 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 period of 4 hours. The results are shown in Figures 9 and 10. Figure 9 shows the data for the vesicles VI, V2 and V3 with intact erythrocytes. The incubation medium contained 10 ml of RBC (red blood cells), 10 ml IHP-loaded vesicles in isotonic 0.1 M bisTris 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 minutes for the V2 vesicles (which show also the highest radioactivity level in the RBC) and 35 minutes for VI and V3. i7329 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) Biochemistry 1_6, 3943 - 3948) that cholesterol exchanges between vesicles and erythrocytes. In the RBC lipid extracts (see Figure 10) we found a τ^2 30 minutes when VZ vesicles were used. When V2 and V3 vesicles are incorporated into Hela cells the same pattern is obtained (see Figure 11). The incubation of the Hela cells with the vesicles was made under the same conditions as described before. In another set of experiments, Hela cells were incubated with 14C-cholesterol-contain!ng vesicles in isotonic buffers, at several pH values between 7 and 8. Figure 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 radioactivity labelled vesicles is the same as the half-life time of the IHP uptake by erythrocytes incubated with dialyzed, IHP-loaded vesicles(Figure 16). This is additional proof that we measure not only lipid exchange between cells and vesicles but fusion of vesicles with the cells.

Thin layer chromatograms of the erythrocyte lipid extract showed the enrichment of the RBC membrane lipids with the lipids of the vesicles.

Apparent pH-optimum of the V2-mediated IHP uptake by stored human red cells.

The uptake of IHP by washed erythrocytes depends on the pH of the incubation medium. Plotting ρθ (1/2) of IHP-loaded erythrocytes versus pH of incubation medium (see Figure 13) demonstrates an apparent pH optimum of IHP incorporation in the range of pH 7.4 to 7.5. The decrease of pn (1/2) above pH 7.5 u2 corresponds to the Bohr effect curve shown in Figure 6 and correlates with the decrease of IHP affinity to haemoglobin. Below pH 7.4 the dramatic decrease of pn (1/2) indicates a limited IHP u2 incorporation and therefore the theoretically expected pn (1/2) for totally transformed haemoglobin by bound IHP is not u2 observed.

The change of buffering medium to pH 7.4 after incubation at pH 7.8 increases the pn (1/2) of intra-erythrocytic haemoglobin 2 to values of 30 to 40 mm Hg at 25°C as demonstrated in Figure 6.

Incubation at pH 7.8 and buffering to different pH-values lead to an increase of pn (1/2) over the whole range of pH. From u2 this result we can conclude that incorporation of IHP into 20 erythrocytes is more effective above pH 7.4 although at lower pH values are also effective.

Kinetics of the IHP uptake by stored human red cells Figure 14 shows the time-dependent decrease of the 02 affinity of erythrocytes after incubation with IHP-loaded V2 vesicles in 0.19 M IHP solution at pH 7.35. The increase of pn (1/2) u2 reaches its half-maximum value after 4 minutes.

The kinetics of IHP incorporation measured as increase of pn (1/2) depends on the u2 *73 39 pH of the incubation medium. In Figure 15 the half-life time of IHP incorporation is plotted versus pH. The uptake of IHP is a slower reaction at low pH (pH 7.3) and a faster reaction at higher pH (pH 7.7). The short half-life time of incorporation at high pH corresponds to the larger amount of IHP incorporated into erythrocytes (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 time of IHP incorporation to 30 minutes at pH 7.4 (see Figure 16).

ATP level in IHP-loaded erythrocytes The adenosine triphosphate (ATP) content of erythrocytes is of great interest from the viewpoint of red cell preservation and of intact function. The ATP level was measured in red cells having 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-luciferase system: E + oxidised substrate + AMP + COg + hv. The reaction is so efficient that one proton is produced for each ATP molecule utilized. The incorporation of empty vesicles or of IHP-loaded vesicles is without significant influence on the ATP level in the erythrocytes. 47333 Table 2; ATP content of erythrocytes.

ATP uM/ml RBC Erythrocytes 0.92 + 10% Erythrocytes-V3 0.84 Erythrocytes-V2-IHP 0.93 The data are for one week-old RBC. The lack of change of the ATP level in the RBC after incorporation of IHP indicates unaltered cell-viability, functionality and plasticity of the RBC.

Qg-releasing effect by fusion of IHP-loaded vesicles _with erythrocytes_ .

The right-shift of the 02-binding curves after incorporation of IHP is shown in Figure 17. After fusion of 41 day old erythrocytes, suspended in isotonic bis-Tris buffer, pH = 7.4, with the IHP-loaded V2 vesicles and 02 half-saturation pressure increases from 7 to 28 mm Hg. This means that the normal but aged erythrocytes are loaded at 25°C to 95% with 02 under an 0,,-partial pressure of 30 mm 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 bound IHP after fusion of the IHP-loaded vesicles with the erythrocytes.

Under physiological conditions (at 37°C) a Og half-saturation pressure of 60 mm Hg is computed for the IHP loaded erythrocytes at pH 7.4. Under a critical Og partial pressure of 30 mm Hg in the brain, 80% of the haemoglobin from vesicle-treated erythrocytes would release the bound Og, while normal, untreated erythrocytes would release under these conditions only 20 to 25% of the oxygen. The effective affinity of the erythrocytes can be varied between these two limits either by varying the IHP concentration in the lipid vesicles or the ratio of treated to untreated erythrocytes in blood.

This result shows that the method which we propose in order to incorporate IHP into the erythrocytes provides a lasting, significant and controlled lowering of the 0g affinity of haemoglobin in intact cells. The erythrocytes thus IHP-loaded are particularly suitable for the control of the Og supply of the tissues in the cases mentioned above.

High-altitude adaptation of rats and dogs.

A 200 g rat (body weight) with a blood volume of 14 ml (pn (1/2) u2 = 14.0 mm Hg at 25°C and pH 7.4) was kept in a chamber under decreasing Og-partial pressure. At an Og-partial pressure of 120 mm Hg equivalent to an altitude of 13200 m the rat tumbled down because of 0g deficiency in the muscles of extremities. Then the 0g pressure in the chamber 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. -4?33» The IHP-loaded erythrocytes were resuspended in the plasma (p^ (1/2) = 28.0 mm Hg at 25°C and pH 7.4) and then retransfused to the rat. After having decreases now the 02-partial pressure the rat tumbled down at 100 mm Hg ( = 14200 m altitude). This treatment therefore caused an increase of the altitute ceiling of + 8%. The altitude adaptation experiment was repeated with this rat 24 hours later and lead to the same result.

In another experiment a 9 kg (body weight) dog with a blood volume of 630 (pn (1/2) = 10.8 mm Hg at 25°C and pH 7.4) was kept in a u2 chamber under decreasing 02-partial pressure. At an 02-partial presure of 140 mm Hg ( = 12200 m altitude) the dog tumbled down.

Then the 0^ pressure in the chamber was quickly restored to the normal value and the dog behaved normally. From this dog 100 ml blood were collected, the erythrocytes were isolated and loaded with IHP as described in Methods. The IHP-loaded erythrocytes were resuspended in the serum (pn (1/2) = 15,0 mm Hg at 25°C and pH 7.4) and then u2 retransfused to the dog. After having decreased the 02-partial pressure the dog tumbled down at 110 Hg ( = 13800 m altitude). Therefore this treatment caused an increase of the altitude 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. *7329 Human erythrocytes with high affinity mutane haemoglobin A 19 year old female patent with a haemoglobin mutant of unknown structure (HbMa-nz) with high Og affinity donated blood. at 25°C pH 7.4 her fresh erythrocytes showed a pn (1/2) u2 = 7.5 mm Hg. Thus the oxygen supply to the tissues is decreased to 50%. Because of oxygen deficienty in her tissues, this patient receives blood transfusions every six weeks. Loading of these erythrocytes with IHP, as described in Methods, led to an increase of ρθ (1/2) = 18.7 mm Hg. This shows that the high Og affinity of this patient's blood can be described 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 total blood exchange.

Claims (14)

1. Modified intact erythrocytes ensuring improved oxygen release of the blood, characterized in that allosteric effectors are irreversibly incorporated into the erythrocytes by means of 5 lipid vesicles and these allosteric effectors are bound to the haemoglobin in the erythrocytes.

2. Modified intact erythrocytes according to claim 1, characterized in that said allosteric effector is inositol hexaphosphate. 10

3. Modified intact erythrocytes according to claim 1, characterized in that said allosteric effector is inositol pentaphosphate.

4. Modified intact erythrocytes according to claims 1 to 3, characterized in that said lipid vesicles are mixtures of phos15 phatidylcholine/phosphatidylserine/cholesterol in a molar ratio of 10 to 5.4 to 1:10 to 3.

5. Modified intact erythrocytes according to claims 1, 2 and 4, characterized in that said allosteric effector is inositol hexaphosphate and said lipid vesicles are a mixture of phos20 phatidylcholine/phosphatidylserine/cholesterol in a molar ratio of 8:2:7.

6. Extracorporal blood or stored blood, characterized in that it contains modified intact erythrocytes according to claims 1 to 5. 25

7. A process for the preparation of modified intact erythrocytes according to claims 1 to 5, characterized in that lipid vesicles loaded with allosteric effectors in carrier fluids are irreversibly incorporated into the interior of erythrocytes and the allosteric effectors are bound to the haemoglobin in the erythrocytes.

8. A process according to claim 7, characterized by (a) dissolving inositol hexaphosphate in an isotonic buffer solution until the solution is saturated, 5 (b) suspending in this solution a lipid mixture containing phosphatidylcholine, phosphatidylserine and cholesterol in the molar ratio 10 to 5:4 to 1:10 to 3, (c) subjecting the resulting suspension to disintegration with ultrasonic waves or an injection process, 10 (d) centrifuging the mixture to separate the supernatent suspension which contains the small inositol hexaphosphate enriched lipid vesicles and free inositol hexaphosphate, thereafter, (e) suspending human erythrocytes having previously been separated from the blood plasma by centrifugation in the supernatent sus15 pension which contains the small inositol hexaphosphate-enriched lipid vesicles and free inositol hexaphosphate, (f) incubating the resulting suspension thereby effecting fusion of the vesicles with the erythrocytes and (g) washing the now modified intact erythrocytes with isotonic 20 NaC1 solution or isotonic buffer and quantitatively freeing them from free inositol hexaphosphate being outside the erythrocytes and suspending them in blood plasma or blood plasma substitute solutions.

9. Compositions for increasing the oxygen release of blood, 25 characterized in that they contain allosteric effectors and mixtures of phosphatidylcholine, phosphatidylserine and cholesterol and acceptable carrier materials and adjuvants.

10. Compositions according to claim 9, characterized in that said allosteric effector is inositol hexaphosphate.

11. Composition according to claims 9 and 10, characterized in that said lipid vesicles are mixtures of phosphatidylcholine, phosphatidyl serine and cholesterol in a molar ratio of 10 to 5:4 to 1:10 to 3. 5

12. Compositions according to claims 9 to 11, characterized in that they contain inositol hexaphosphate and a mixture of phosphatidylcholine, phosphatidyl serine and cholesterol in a molar ratio of 8:2:7.

13. A process for the preparation of modified intact erythro 10 cytes substantially as described herein with reference to the accompanying drawings.

14. Product of a process according to any one of claims 7, 8 or 13.