STROMA-FREE HEMOGLOBIN SOLUTION, PROCESS FOR THE PREPARATION THEREOF AND ITS APPLICATION

Abstract: Hemoglobin solution is stabilized by adding inositol hexaphosphate to give a molar ratio IHP/HB of from 0.1:1 to 1:1 by preference reduced NAD is also added. The stabilized solution is I.A. suitable for calibrating blood-gas control apparatus.

Description: STROMA-FREE H'EMOGLOBIN SOLUTION, PROCESS FOR THE PREPARATION THEREOF AND APPLICATION THEREOF. THIS INVENTiON, MADE BY DRS. R. SPROKHOLT AND DR. .H.J. MAAS, RELATES TO A STABLE STROMA-FREE HEMOGLOBIN SOLUTION, A PROCESS FOR THE PREPARATION THEREOF ANG APPLICATION OF SAID SOLUTION AS STANDARD FOR CALIBRATION AND QUALITY CONTROL OF BLOOD-GAS CONTROL APPARATUS AND MEASURING HEMOGLOBIN-DERIVATIVES (HB-DERIVATIVES). STROMA-FREE HEMOGLOK N SOLUTIONS AND A PROCESS FOR THE PREPARATION THEREOF ARE KNOWN, I.A. FROM U.S. PATENT SPECIFICATION 4,401,652. SUCH SOLUTIONS ARE SUITABLE AS BLOOD SUBSTITUTES DURING ACUTE BLOOG SHORTAGE. BUT ARE ESPECIALLY USED AS STANDARD FOR CALIBRATION AND THE QUALITY CONTROL OF DETERMINATIONS OF PH, PRO2, P 02 AND HB DERIVATIVES IN BLOOD. DETERMINATIONS OF PH1 PLOT AND P02 AND HEMOGLOBIN DERIVATIVES IN BLOOD ARE PRESENTLY DONE QUITE FREQUENTLY IN CLINICAL AND OTHER LABORATORIES. THE THREE FIRST MENTIONED DETERMINATIONS GENERALLY ARE DONE BY MEANS OF SPECIFIC ELECTRODES AND ARE ALWAYS RELATIVE, THAT IS TO SAY THAT THE UNKNOWN SAMPLE IS COMPARED WITH A KNOWN SOLUTION. SIMILARLY THE CONTENTS OF HEMOGLOBIN (HB) AND HB DERIVATIVES ARE GENERALLY DETERMINED SPECTROPHOTO METRICALLLY. THE QUALITY OF THE COMPARATIVE COMPOSITION THIS IS OF PREDOMINATING IMPORTANCE. SAID CALIBRATION WITH STANDARD SOLUTIONS AND THE QUALITY CONTROL THEREOF SHOULD BE DONE EVERY DAY (AND IN THE UNITED STATED OF NORTH-AMERICA IT WAS ALREADY SUGGESTED TO MAKE THIS LEGALLY REQUIRED) AND PREFERABLY THREE SOLUTIONS ARE CALIBRATED, ON HAVING THE RELEVANT PARAMETERS AT OR ABOUT THE NORMAL VALUE FOR HEALTHY PERSONS, ONE WITH SAID PARAMETERS NEAR THE LOWEST VALUE OCCURING IN PRACTICE, AND ONE WITH SAID PARAMETERS NEAR THE HIGHEST VALUE OCCURRING IN PRACTICE. AN INCREASED OR REDUCED ACIDITY IS ACCOMPANIED BY INCREASED OR REDUCED PCO2 AND P02 RESPECTIVELY. DESIRABLE ARE CONTROL COMPOSITIONS HAVING PH OF RESPECTIVELY 7,2, 7,4 AND 7,6 HAVING A P02 OF RESPECTIVELY ABOUT 130. 100 AND 70 MMHG, AND A PLOT 2 OF RESPECTIVELY ABOUT 60, 40 AND 20 MMHG. ALTHOUGH IT IS IN PRINCIPLE GENERALLY KNOWN TO CHECK A CONTROL APPARATUS BY INTRODUCING A KNOWN COMPOSITION THEREIN, THIS CAUSES GREAT PROBLEMS WITH BLOOD. BLOOD IS LIABLE TO METABOLISM SO THAT THEPH, PCO2,PO2 AND THE FRACTIONS OF THE HB-DERIVATIVES CHANGE. MOREOVER GAS EXCHANGE WITH THE SURROUNDINGS MAY APPEAR EASILY, AS THE PARTIAL PRESSURES OF CARBON DIOXIDE AND OXYGEN DEVIATE STRONGLY FROM THOSE IN THE BLOOD. THUS EVERY DAY A NEW STANDARD COMPOSITION SHOULD BE TAKEN FROM A CLOSED VESSEL. AFTER EQUILIBRATING OR TONOMETERING THEREOF WITH A SELECTED GAS MIXTURE (MOSTLY THESE ARE THREE DIFFERENT MIXTURES) THE DESIRED VALUES ARE OBTAINED. FOR THOSE ACQUAINTED WITH TONOMETERY THIS WILL BE CLEAR. AND THE KEEPABILITY OF SAID STANDARD COMPOSITION SHOULD MEET VERY HIGH REQUIREMENTS. THE PH SHOULD BE WITHIN 0.01 OF THE INDICATED VALUE, AND THE PC02 AND P02 2 WITHIN 1MMHG OF THE VALUES GIVEN THEREFOR. AND THIS SHOULD APPLY DURING THE GUARANTEED SHELF-LIFE (UNTIL THE OF THE EXPIRATION DATE) BLOOD, WHICH IS NEITHER CONSERVABLE NOR HOMOGENEOUS, IS UNSUITABLE AS CALIBRATING AND REFERENCE LIQUID. HEMOGLOBIN SOLUTIONS, WHICH ARE OBTAINED BY HEMOLYSIS OF RED BLOOD CORPUSCLES (ERYTHROCYTES) AND CENTRIFUGING OF THE DEBRIS AND OTHER UNDISSOLVED MATERIAL (THE STROMA) DO BETTER. ,PREPARATION METHODS OF STROMA-FREE HEMOGLOBIN SOLUTIONS ARE DESCRIBED IN THE EAST-GERMAN PATENT SPECIFICATION 150,543 AND THE ABOVE-MENTIONED U.S. PATENT SPECIFICATION 4,401,652. LTHOUGH SUCH COMPOSITIONS ARE HOMOGENEOUS AND CONSEQUENTLY ALREADY MUCH BETTER THEY ARE NOT STABLE EITHER. EVEN IF BACTERIAL INFECTION IS PREVENTED BY ASEPSIS THE ACTIVE HOMOGLOBIN CONTENT DECREASES RATHER QUICKLY, MAINLY IN THAT HEMOGLOBIN CONVERTS INTO (INACTIVE) METHEMOGLOBIN (AN OXIDATION OF FE(II) INTO FE(III). THE AFFINITY OF METHOMOGLOBIN FOR OXYGEN IS SUBSTANTIALLY ZERO. THEREFORE FOR CALIBRATION AND QUALITY CONTROL OF BLOOD GAS CONTROL APPARATUS AND METERS OF HB DERIVATIVES GENERALLY USE MADE OF AQUEOUS BUFFER SOLUTIONS OR FLUOROHYDROCARBON SOLUTIONS ALSO CONTAINING A BUFFER AND MOREOVER SUCH AN AMOUNT OF BICARBONATE THAT WITH THE PREVAILING PH ALSO THE CORRECT PCO2 PREVAILS;THEY CAN ABSORB REVERSIBLY CONSIDERABLE AMOUNTS OF OXYGEN. FOR THE DETERMINATION OF THE HB CONTENTS A GOOD CALIBRATION LIQUID BASED ON HICN IS AVAILABLE, BUT FOR THE QUALITY CONTROL OF THE DETERMINATION METHOD IT FAILS. EMULSIONS OF FLUOROHYDROCARBONS FOR SUBSTITUTING BLOOD IN THE CALIBRATION AND CONTROL OF THE QUALITY OF BLOOD CONTROL APPARATUS ARE l.A. DESCRIBED IN U.S. PATENT SPECIFICATION54.299,728 AND 4,369,127. SUITABLE FLUORO HYDROCARBONS ARE PERFLUOROMETHYLCYCLOHEXANE. PERFLUORODIMETHYLDECALIN AND MIXTURES THEREOF AND THE PERFLUOROTRIBUTYLAMINE. I.A. THE COURSE OF THE P02 WITH THE AMOUNT OF OXYGEN ABSORBED IS QUITE DIFFERENT FROM WITH HEMOGLOBIN. AS IS KNOWN WITH A HEMOGLOBIN SOLUTION THE PLOT OF THE 02-TENSION AGAINST THE AMOUNT OF ABSORBED OXYGEN SHOWS AN S FORM, BUT IT IS LINEAR WITH OTHER OXYGEN-BUFFERS SUCH AS THE FLUOROHYDROCARBONS. WITH HEMOGLOBIN THE INCLINATION IS MUCH STEEPER THAN WITH OTHER KNOWN SUBSTANCES, WHICH IS DUE TO CONFORMATION CHANGES WHEN OXYGEN IS ABSORBED. THERE WAS NO LACK OF EFFORTS TO MAKE HEMOGLOBIN SOLUTIONS STABLE. NATURALLY HEMOGLOBIN SEENS TO BE ASSOCIATED WITH 2,3-DIPHOSPHOGLYCERATE (DPG) WHICH HAS SOME STABILIZING INFLUENCE ON HEMOGLOBIN. BUT SAID COMBINATION DISSOCIATES VERY EASILY AND ITS STABILITY IS INSUFFICIENT FOR PRACTICE. IN GERMAN AUSLEGESCHRIFT 2,617,822 IT IS PROPOSED TO USE FOR THIS AIM 1,1 G OF PYRIDOXALPHOSPHATE PER LITER OF 6-8 % HEMOGLOBIN SOLUTION, WHICH HOWEVER APPEARS TO BIND WITH THE HEMOGLOBIN (AS A RESULT OF THE ALDEHYDE GROUP) TO RESULT IN A COMBINATION A PART OF WHICH HAS A CERTAIN AFFINITY FOR OXYGEN AND THE OTHER PART A DIFFERENT, LOWER AFFINITY. THUS THERE ARE IN SUCH A COMPOSITION VIRTUALLY TWO KINDS OF HEMOGLOBIN, AND THE PROCEEDING OF THE PLOT OF THE ABSORBED OXYGEN AGAINST THE P02 IS THEREFORE IS CONSIDERABLY SMOOTHER, WHICH IS UNFAVOURABLE FOR A CALIBRATION LIQUID AND A COMPOSITION FOR QUALITY CONTROL. ANOTHER HEMOGLOBIN COMPOSITION CONSITS ON THE ONE HAND OF LYOPHILISED HEMOGLOBIN AND ON THE OTHER HAND A BUFFER SOLUTION HAVING ALL REMAINING NECESSARY COMPONENTS, WHICH ARE TO BE MIXED JUST BEFORE USING THEM. SUCH A COMPOSITION HAS THE USUAL DISADVANTAGE THAT THE DISSOLUTION SHOULD PROCEED VERY COMPLETELY, OTHERWISE DOUBT REMAINS. MOREOVER DRY HEMOGLOBIN (DIFFERENT FROM HEMOGLOBIN IN SOLUTION) IS EXTREMELY SENSITIVE FOR OXIDATION INTO METHEMOGLOBIN, AS IS DESCRIBED BY DEVENUTO ET AL. IN SURG. GYNEC. OBST. 48 (1979) 69-75 AND BY LABRUDE ET AL IN J. PHARM. PHARMACOL. 33 (1981) 115-117. NOW IT WAS FOUND THAT THE ABOVE OBJECTIONS ARE OVERCOME AND A STABLE COMPOSITION WILL BE HAD, WHICH IS WELL SUITABLE UNDER ALL CIRCUMSTANCES, IF HEMOGLOBIN(HB) IS PROTECTED WITH A LIMITED AMOUNT OF INOSITOL HEXAPHOSPHATE (IPH). THEREFORE A STROMA-FREE HEMOGLOBIN SOLUTION ACCORDING TO THE INVENTION CONTAINS INOSITOL HEXAPHOSPHATE IN SUCH AN AMOUNT THAT THE MOLAR RATIO IHP/HB IS FROM 0,1:1 TO 1,0:1. IT APPEARED THAT IN SUCH A SOLUTION IMMEDIATELY AFTER MIXING LESS THAN 2 % OF THE TOTAL AMOUNT OF HEMOGLOBIN IS METHEMOGLOBIN AND, AFTER KEEPING IT AT 40C OR LOWER DURING ONE YEAR, LESS THAN 7 %. THE P50 (I.E. THE OXYGEN TENSION WITH 50 , LOAD OF THE TOTAL HEMOGLOBIN WITH OXYGEN) DECREASES SOMEWHAT IN THAT METHEMOGLOBIN HAS AN EXTREMELY LOW P50. IF 5 % OF THE HEMOGLOBIN CONVERTS INTO METHEMOGLOBIN THE P50 DECREASES WITH 0.5 MMHG (67 PA). IN ANY CASE THE P50 OF THE COMPOSITION WHEN KEEPING IT IN THE REFRIGERATION WILL DECREASE IN SIX MONTHS NO MORE THAN 2 MMHG (267 PA). A COMBINATION OF HEMOGLOBIN WITH INOSITOL HEXOPHOSPHATE WAS, AS FAR AS WE ARE AWARE OF, ONLY MENTIONED BEFORE IN AN ARTICLE BY P.M. BREEPOEL ET AL IN PFLUGERS ARCHIV 389 (1981) 227-235, WHEREIN IT IS SHOWN THAT IHP AS WELL AS DGP AND ATP (ADENOSINTRIPHOSPHATE) REDUCES THE OXYGEN AFFINITY OF BOVINE HEMOGLOBIN. HOWEVER THE DIFFERENCES WERE SMALL, AND THE KEEPABILITY OF SIMILAR COMBINATIONS HAD NOT BEEN EXAMINED. THE MOLAR RATION IHP/HB OF THE SOLUTION DESCRIBED IN SAID ARTICLE WAS 2:1. FURTHERMORE IT WAS FOUND THAT THE CONVERSION OF HEMOGLOBIN INTO METHEMOGLOBIN, ALREADY LOW, CAN BE RETARDED FURTHER BY USING THE ENZYME METHEMOGLOBIN REDUCTASE, WHICH OCCURS NATURALLY IN ERYTHROCYTES AND CONSEQUENTLY IN THE HEMOGLOBIN SOLUTION ACCORDING TO THE INVENTION. SAID ENZYME REVERTS THE IRREVERSIBLE CONVERSION OF HEMOGLOBIN + METHEMOGLOBIN. REQUIRED THEREFORE IS THE PRESENCE OF NADH (REDUCED FORM OF NICOTINE ADENINE DINUCLEOTIDE) OF WHICH SO MUCH HAS TO BE PRESENT THAT THE MOLAR RATIO NADH/HB IS FROM 0.25;1 TO 1,50 1, OPTIMAL IS A MOLAR RATIO NADH/HB OF ABOUT 0.5:1. INSTEAD OF NADH ALSO NADPH (THE REDUCED FORM OF NICOTINE ADENINE DINUCLEOTIDE PHOSPHATE) MAY BE PRESENT. THE HEMOGLOBIN CONCENTRATION OF THE COMPOSITION WILL BE GENERALLY IN THE RANGE FROM 3 MMOL/L TO 15 MMOL/L; PREFERABLY IT IS ABOUT 8 MMOL/L. THE CONTENTS OF SODIUM. POTASSIUM, CHLORIDE AND OPTIONALLY OTHER IONS WILL BE WITHIN THE PHYSIOLOGICAL RANGES. THE BICARBONATE CONTENT WILL BE DETERMINED BY THE DESIRED PH AND THE DESIRED PC02. IN OXYGEN-BUFFERNG CAPACITY SAID COMPOSITION IS EQUAL TO BLOOD, AND THE P50 REACTS TO A SHIFT OF THE PH IN ABOUT THE SAME WAY AS OF BLOOD, IF ANY. THE P50 IS ALSO SENSITIVE TO THE IHP CONTENT. HEREWITH THE PSO CAN BE ADJUSTED AT NORMAL AT THOSE ACID-BASE CONDITIONS THAT ARE CHARACTERIZING FOR ACIDOSIS, NORMAL CONDITION AND ALKALOSIS. IT IS ALSO PREFERABLE, AS IS INDICATED ABOVE, TO PROVIDE THE CALIBRATION AND REFERENCE LIQUIDS ACCORDING TO THE INVENTION IN SERIES OF THREE, ONE WITH PH, PLOT AND P02 2 AT OR ABOUT THE NORMAL VALUES FOR HEALTHY PERSONS, ONE WITH SAID PARAMETERS NEAR THE LOWEST VALUE OCCURRING IN PRACTICE, AND ONE WITH SAID PARAMETERS NEAR THE HIGHEST VALUE OCCURRING IN PRACTICE. THE INVENTION ALSO RELATES TO A PROCESS FOR PREPARING A STROMA-FREE HEMOGLOBIN SOLUTION. THEREFOR BEFORE HEMOLYSIS THE ERYTROCYTES ARE PURIFIED BY REPEATED RINSING WITH AN ISOTONIC OR WEAKLY HYPERTONIC NACL SOLU TION AND PREFERABLY THEY ARE ALSO PREVIOUSLY STRIPPED OF LEUCOCYTES BY FEEDING A SUSPENSION THEREOF THROUGH A COLUMN WITH A SUITABLE ABSORBENT (E.G. ERYPUR OF THE ORGANON COMPANY). THIS WAS KNOWN I.A. FROM THE ABOVE MENTIONED U.S. PATENT SPECIFICATION. THE LYSIS IS REALISED BY BRINGING THE PURIFIED ERYTROCYTES INTO A MINIMUM OF TWO VOLUMES BUT PREFERABLY IN FOUR VOLUMES OF A STRONGLY HYPOTONIC SOLUTION, PREFERABLY STERILE DISTILLED OR DEMINERALIZED WATER, WHICH, AS PRECAUTION1 GENERALLY ALSO CONTAINS SOME BACTERICIDE. IF DESIRED THE LYSIS CAN BE SPEEDED UP WITH ULTRASONIC VIBRATIONS BUT THIS IS NOT ESSENTIAL. PREFERABLY THE LYSATE IS CENTRIFUGED, SO THAT THE STROMA (DEBRIS OF THE ERYTHROCYTE CELL WALLS AND OTHER SOLIDS) IS REMOVED. THE SUPERNATANT IS EVACUATED CAREFULLY FROM THE SEDIMENT BY MEANS OF A SIPHON. IF DESIRED SAID LYSATE IS CONCENTRATED, WHICH E.G. CAN BE DONE BY EVAPORATION BUT PREFERABLY BY ULTRAFILTRATION, E.G. BY MEANS OF SHAFTS OF HOLLOW FIBRES. TO SAID SOLUTION THE IONS NA+, K+, CA 2+, Mug 2+1 HCO AND 3 CL ARE ADDED AS SALTS, IN SUCH AMOUNTS THAT THE FINAL SOLUTION WILL HAVE THE DESIRED CONCENTRATIONS OF SODIUM, POTASSIUM, MAGNESIUM AND CHLORIDE. BY PREVIOUS CAREFUL CALCULATION THE AMOUNT OF BICARBONATE THE PH WILL THEN BE AT THE DESIRED VALUE. OFTEN IT WILL BE NECESSARY TO CENTRIFUGE THE SOLUTION ONCE AGAIN TO OBTAIN A REALLY CLEAR FINAL COMPOSITION. FINALLY BY GASSING WITH A MIXTURE OF NITROGEN, CARBON DIOXIDE AND OXYGEN THE PCO2 AND P02 ARE ADJUSTED AT THE CORRECT VALUES. THE PROCESS OF THE INVENTION IS NOW CHARACTERIZED IN THAT AFTER THE LYSIS SO MUCH INOSITOL HEXAPHOSPHATE IS ADDED THAT THE MOLAR RATIO IHP/HB IS FROM 0,1:1 TO 1,0:1. THE YIELD OF HEMOGLOBIN MAY BE AS MUCH AS 90-95 %. IT APPEARED THAT WITH SAJD PREPARATION OPTIMALLY TOGETHER WITH THE IHP ALSO NADH IS BE ADDED, THE BEST AT SUCH A CONCENTRATION THAT THE MOLAR RATIO NADH/HB BECOMES ABOUT 0.5:1. MOREOVER IT APPEARED THAT THE STABILITY, ALREADY HIGH, OF THE COMPOSITION MAY BE INCREASED FURTHER, SUCH THAT EVEN AFTER 1 YEAR AT 40C OR LOWER ABSOLUTELY NO PRECIPITATION IS FOUND IN IT. IF THE HEMOGLOBIN SOLUTION IS SUBJECTED TO A DOUBLE PH SHIFT AND THE PRECEPITATES THEN APPEARING IN IT ARE SEPARATED BEFORE THE SOLUTION IS BROUGHT AT THE DESIRED FINAL CONTENTS OF HB, IHP, NADH AND OTHER COMPONENTS AND THE DESIRED 2 AND CO2 TENSIONS ARE ADJUSTED ACCORDING TO THE INVENTION SO MUCH IHP IS ADDED TO THE LYSATE THAT THE MOLAR RATIO OF IHP/rIB IS ABOUT 0.5:1. WITH DILUTED NAOH THE PH IS ADJUSTED BETWEEN 8,0 AND 8,1. THE PRECIPITATE IS REMOVED BY CENTRIFUGING. THE PH IS NOW ADJUSTED WITH DILUTED HCL AT BETWEEN 6,9 AND 7,0. THE PRECIPITATE IS REMOVED BY CENTRIFUGING AND FINALLY AFTER ADDING SODIUM, POTASSIUM, CALCIUM3 MAGNESIUM, BICARBONATE AND CHLORIDE TO ATTAIN THE DESIRED VALUES THE PH1 PCO2 AND PO ARE ADJUSTED. FINALLY THE INVENTION ALSO RELATES TO THE APPLICATION OF SAID STABLE COMPOSITION. IN THE FIRST PLACE IT MAY BE USED WHEN DETERMINING THE PH, PC02, PO2 ANDOR HEMOGLOBIN CONTENT IN BLOODy THIS DETERMINATION NOW BEING CALIBRATED WITH A SOLUTION AS DESCRIBED ABOVE. AS IS ALREADY SAID THIS HAS THE ADVANTAGES THAT ALSO AFTER KEEPING IT FOR A LONG TIME AN ABSOLUTELY RELIABLE COMPOSITION IS OBTAINED WHICH CAN BE USED WITHOUT ANY PRECAUTION AND WHICH IS ALSO FREE OF HAZE. BUT SUCH A HEMOGLOBIN SOLUTION MAY ALSO BE USED AS BLOOD SUBSTITUTE HAVING THE ADVANTAGE THAT IT DOES NOT SHOW IMMUNOGENITY (BY THE ABSENCE OG THE ERYTHROCYTE CELL WALLS). ON THE OTHER HAND IT WILL BE EXCRETED VERY QUICKLY IN THAT THE KIDNEYS DO NOT RETAIN AT ALL HEMOGLOBIN NOT LOCKED WITHIN THE RED BLOOD CORPUSCLES. THUS IT IS SPECIALLY CONSIDERED FOR INFUSION IN EMERGENCY CASES. FURTHER IT MAY ALSO BE USED AS STANDARD FOR THE (SPECTROPHOTOMETRICAL) DETERMINATION OF HEMOGLOBIN AND HB DERIVATIVES. THE INVENTION IS NOW ELUCIDATED BY THE FOLLOWING EXAMPLES. EXAMPLE 1 ALL ACTIONS WERE DONE UNDER STERILE CONDITIONS (LAMINAIR AIRFLOW CABINET, CLASS 100;STERILE SOLUTIONS, ETC.). 1) HUMAN BLOOD WAS COLLECTED IN ACD SOLUTION, PORTIONS OF 420 ML IN BOTTLES OF 500 ML IN WHICH ALREADY 80ML OF ACD SOLUTION WAS PRESENT. TOTALLY 6 LITERS. 2) SAID BOTTLES WERE PLACED IN CENTRIFUGE CUPS AND CENTRI FUGED AT 3000 X G DURING 30 MINUTES. THE SUPERNATANTS WERE EVACUATED THROUGH SIPHONS. PER BOTTLE 200-250 ML OF CELLS WERE RECOVERED. 3) THE BLOOD CELLS WERE SUSPENDED IN Or9 % NACL SOLUTION, FINAL VOLUME 500 ML PER BOTTLE. 4) THE BLOOD CELL SUSPENSIONS WERE FED THROUGH A COLUMN LEUCOFILTER (ERYPUR OF THE ORGANOM COMPANY) SO THAT THE WHITE BLOOD CELLS AND A GREATER PART OF THE BLOOD PLATELETS WERE REMOVED. 5) THE BLOOD CELL SUSPENSIONS WERE CENTRIFUGED AT 3000 X G DURING 30 MINUTES. THE SUPERNATANTS WERE REMOVED WITH A SIPHON. 6) SIMILARLY TO (3) MAKING UP TO 500 ML PER BOTTLE WAS DONE WITH 0.9 % NACL SOLUTION. 7) SIMILARLY TO (5) CENTRIFUGING WAS DONE AGAIN AT 3000 G DURING 30 MINUTES1 THE SUPERNATANTS WERE DISCARDED. 8) PER BOTTLE THERE WERE NOW ABOUT 200 ML OF ERYTHROCYTES. 9) ALL PORTIONS OF ERYTHROCYTES WERE COMBINED AND MIXED WELL, AND SUBSEQUENTLY DISTRIBUTED OVER BOTTLES OF 3 LITERS, EACH BOTTLE CONTAINING 0.6 LITER OF ERYTHROCYTES. NOW TO EACH BOTTLE 2.4 LITERS OF STERILE1 DISTILLED WATER WAS ADDED AND MIXED. ALSO PER LITER 1 G OF NEOMYCIN SULPHATE (DISSOLVED IN ABOUT 2 ML WATER) WAS ADDED (TO PREVENT BACTERIAL GROWTH) . SAID MIXTURES WERE KEPT IN A REFRIGERATOR AT +40C DURING ONE NIGHT. 10) THE COOLED HEMOGLOBIN SOLUTION WAS DISTRIBUTED AT ROOM TEMPERATURE OVER POLYCARBONATE CENTRIFUGE BOTTLES (STERILE1 WITH A SPECIAL CLOSURE). HEREIN THEY WERE CENTRIFUGED AT 0-100C AT 27,000 G IN A SORVALL RC-5 CENTRIFUGE WITH GSA-ROTOR DURING 30 MINUTES. 11) THE ABOVE LIQUIDS WERE STERILELY EVACUATED AND COLLECTED IN BOTTLES OF 3 LITERS. 12) HERETO CACL2 AND NACL WERE ADDED AT CONCENTRATIONS OF RESP. 0.02 M AND 0.13 M AND SUBSEQUENTLY 8 ML OF 0.25 M SODIUM INOSITOL HEXAPHOSPHATE SOLUTION WERE ADDED TO 3 LITERS, THE PH OF WHICH BEING ADJUSTED AT 8,0 WITH DILUTE HCL, SO THAT IHP/HB WAS NOW 0.5:1. NOW 1 M NAOH WAS ADDED UNTIL THE PH WAS 9.05. A PRECIPITATE FORMED (CA-IHP, MEMBRANE PROTEINS). THE IHP-NA-SALT HAD BEEN PURCHASED FROM SIGMA CHEMICALS (ST. LOUIS, MO, 63178, USA) 13) IN A SORVALL CENTRIFUGE CENTRIFUGING WAS DONE AT 27,000 G DURING 20 MINUTES. 14) THE ABOVE HEMOGLOBIN SOLUTIONS WERE CAREFULLY EVACUATED AND COMBINED. 15) WITH 1 M HCL THE PH WAS ADJUSTED AT 7,0. AGAIN A PRECIPITATE FORMED (PROTEIN). 16) AGAIN CENTRIFUGING WAS DONE AT 27,000 G DURING 30 MINUTES. 17) THE ABOVE HEMOGLOBIN SOLUTIONS WERE EVACUATED AND COMBINED. 18) pREVIOUS TO CONCENTRATING THE SOLUTION WAS NOW FILTERED THROUGH PRE-FILTERS AND ULTRA-FILTERS HAVING PORES OF 0.8us. 19) THE SOLUTION WAS CONCENTRATED BY ULTRA-FILTRATION BY MEANS OF SHAFTS OF HOLLOW FIBRES (APPARATUS AMICON DC-2 WITH H1P10-8 CARTRIDGES). THIS WAS CONTINUED UNTIL A HEMOGLOBIN CONCENTRATION OF 10 M WAS ATTAINED. -0) NOW NADH IN WATER WAS ADDED, ABOUT 7 G IN TOTAL, SO T THE MOLAR RATIO NADHHB WAS NOW 0,5:1. 21) NOW HB, PH, PRO2, NA, K1 CL AND FREE-IONISED CALCIUM DETERMINED DIRECTLY MEASURED AND THE PH1 PCO2 AND THE BOCARBONATE CONTENT WERE DETERMINED WITH THE ASTRUP TECHNIQUE. 22) THE OBTAINED LIQUID WAS DIVIDED INTO THREE ROUGHLY EQUAL PARTS. FROM THE RESULTS OF THE DETERMINATIONS (21) IT WAS CALCULATED HOW MUCH OF THE VARIOUS IONS SHOULD BE ADDED TO OBTAIN A COMPOSITION AS NEAR AS POSSIBLE TO THAT OF BLOOD PLASMA. 23) NOW NACL, KCL, CACL2 AND NAHCO3 WERE ADDED, SO THAT IN EACH PORTION THE RELATION BETWEEN PH AND PRO 2 WAS KNOWN. (PBAR = 760 MMHG) PH PCO2 = 101,3 KPA0 7.20 60 MMHG (8.00 KPA) 7.40 40 MMHG (5.33 KPA) 7.60 20 MMHG (2.67 KPA) 24) TO EACH PORTION SO MUCH IHP WAS ADDED THAT FOR EACH PH/PCO2 COMBINATION THE P50 WAS ABOUT 26.5 MMHG (3.53 KPA) (FOR THIS PURPOSE ADD THEE DIFFERENT AMOUNTS OF IHP AND DETERMINE THE P02 AT A CONSTANT PCO2, WHEREAFTER BY INTRAPOLATION THE DESIRED IHP CONCENTRATION IS FOUND; P02 IS A FUNCTION OFHIHP) 25) THEN CENTRIFUGING WAS DONE AGAIN IN A SORVALL CENTRI FUGE AT 27,Q00 G DURING 20 MINUTES. 26) THE ABOVE HEMOGLOBIN SOLUTION WAS EVACUATED AND STERILIZED BY FILTRATION THROUGH AN ULTRA-FILTER HAVING PORES OF 0.22 pM. 27) THE PRODUCT WAS STERILELY DISTRIBUTED OVER BOTTLES WITH RUBBER LUGS. THE PRODUCT CONTAINES 8,0.10 M OF HB, OF WHICH LESS THAN 2 % WAS METHEMOGLOBIN (AFTER 1 YEAR IN THE REFRIGERATOR LESS THAN 7 /a). THE HEMOGLOBIN YIELD IN THIS CASE WAS ABOUT 60 S. IT CONTAINED 0.21 M OF NA; 0.011 M OF K AND 0.16 M OF CL'. IT CONTAINED EXTREMELY FEW DISCRETE PARTICLES, WHICH ANYHOW WERE SMALLER THAN 0.22 eM. AFTER 251-FOLD DILUTED WITH HICN REAGENT IT WAS FOUND; 1 CM E750 N; 0.0002 AND E540lE514= 1.59. THE RELATION BETWEEN P50 AND PH WAS THE SAME AS IS KNOWN FROM THE LITERATURE, AND THE TEMPERATURE COEFFICIENTS FOR PH, PCO2 AND P02 WERE APROXIMATELY THOSE OF BLOOD. EXAMPLE II 1) BOVINE BLOOD WAS MADE NON-CLOTTING WITH A HEPARIN SOLUTION (550 ML OF BLOOD + 3 ML OF SOLUTION). 2) - 11) EXACTLY AS WITH EXAMPLE I. 12) TO THE HEMOGLOBIN SOLUTION CACL2 AND NACL WERE ADDED AT CONCENTRATIONS OF RESP. 0.02 M AND 0.13 M AND SUBSEQUENTLY IN TOTAL ABOUT 3.5 ML OF 0.25 M NA-IHP THE PH OF WHICH WAS FIRST ADJUSTED AT 8.0 WITH DILUTE HCL. THE PH OF THIS MIXTURE WAS 7.4. 13) A SLIGHT HAZE WAS REMOVED BY CENTRIFUGING AT 27,000 C DURING 20 MINUTES. 14) - 24) WERE EXACTLY AS (17) - (27) OF EXAMPLE I. THE PROPERTIES OF THIS COMPOSITION WERE SUBSTANTIALLY EQUAL TO THOSE OF THE COMPOSITION OF EXAMPLE I. ONLY AFTER KEEPING IN THE REFRIGERATOR FOR HALF A YEAR IT HAD A VERY SLIGHT HAZE. EXAMPLE III 1) BOVINE BLOOD WAS MADE NON-CLOTTING WITH A HEPARIN SOLUTION (550 ML OF BLOOD + 3 ML OF SOLUTION). 2) - 27) IN ACCORDANCE WITH EXAMPLE I. THE PROPERTIES OF SAID COMPOSITION WARE SUBSTANTIALLY EQUAL TO THOSE OF THE COMPOSITION OF EXAMPLE 1. ALSO THAT EVEN AFTER HALF A YEAR IT DID NOT SHOW ANY TRACE OF HAZE.

Claims: CLAIMS 1. STROMA-FREE HEMOGLOBIN SOLUTION, CHARACTERIZED IN THAT IT CONTAINS INOSITOL HEXAPHOSPHATE, IN SUCH AN AMOUNT THAT THE MOLAR RATIO IHP/HB IS FROM 0.1:1 TO 1.0:1. 2. STOMA-FREE HEMOGLOBIN-SOLUTION ACCORDING TO CLAIM 1 CHARACTERIZED IN THAT IT ALSO CONTAINS METHEMOGLOBIN REDUCTASE AND REDUCED NAD (NADH). 3. STROMA-FREE HEMOGLOBIN SOLUTION ACCORDING TO CLAIM 2, CHARACTERIZED IN THAT IT ALSO CONTAINS CYCTOCHROME B5. 4. STROMA-FREE HEMOGLOBIN SOLUTION ACCORDING TO ANY OF THE PRECEDING CLAIMS, CHARACTERIZED IN THAT IT CONTAINS FROM 3 TO 15 MMOL/L HB AND HAS A PH FROM 6,9 TO 8 , 1. 5. STROMA-FREE HEMOGLOBIN SOLUTION ACCORDING TO ANY OF THE PRECEDING CLAIMS, CHARACTERIZED IN THAT AFTER BEING KEPT FOR 1 YEAR AT BELOW 40C IT STILL CONTAINS NO PRECIPITATE. 6. STROMA-FREE HEMOGLOBIN COMPOSITION ACCORDING TO ANY OF THE PRECEDING CLAIMS CONSISTING OF THREE SOLUTIONS. ll-rH A PH OF ABOUT 7.2, ABOUT 7.4 AND ABOUT 7.6. 7 PROCESS FOR PREPARING A STROMA-FREE HEMOGLOBIN SOLUTION, WHEREIN PREVIOUSLY PURIFIED ERYTHOCYTES ARE LYSED BY ADDING DISTILLED OR DEMINERALIZED WATER AND IN THE LYSATE THE CONTENTS OF CA; NA; K; HCO3 AND CL' ARE ADJUSTED AT THE VALUES CORRESPONDING WITH THOSE OF BLOOD PLASMA, BY GASSING THE PCO2 AND P02 ARE ADJUSTED AT THE DESIRED VALUES, AND THE FINAL COMPOSI TION TS STERILIZED BY FILTERING, CHARACTERIZED IN THAT MOREOVER SO MULL INOSITOL HEXAPHOSPHATE IS ADDED THAT THE MOLAR RATIO IHP/HB IS FROM 0.1:1 TO 1.0:1. 8. PROCESS ACCORDING TO CLAIM 7, CHARACTERIZED IN THAT TO THE LYSAT± SO MUCH IHP IS ADDED, THAT THE MOLAR RATIO IHP/HB IS ABOUT 0.5:1, THE PH IS ADJUSTED BETWEEN 8.0 AND 8.1 WITH DILUTED NAOH, THE PRECIPITATE IS REMOVED BY CENTRIFUGING. THE PH ADJUSTED BETWEEN 6.9 AND 7.0 WITH DILUTED HCL, THE PRECIPITATE IS REMOVED BY CENTRIFUGING, AND FINALLY AFTER ADDITION OF CA; K , NA', HCO3 AND CL' TO ATTAIN THE DESIRED CONTENTS, THE PH AND PCO2 AND P02 ARE ADJUSTED AT THE DESIRED VALUES. 9. PROCESS FOR DETERMINING PH, PCO2, PO2 AND/OR HB IN BLOOD, CHARACTERIZED IN THAT SAID DETERMINATION IS CALIBRATED WITH A SOLUTION OBTAINED ACCORDING TO ANY OF CLAIMS 1-6. 10. APPLICATION OF A COMPOSITION ACCORDING TO ANY OF CLAIMS 1-6 AS BLOOD SUBSTITUTE.

Inventors: MAAS, ANTONIUS HENRICUS JOSEPHUS, SPROKHOLT, RONALD
Applicants: EURO-TROL B.V.
Application year: 1985
Main classification: C07K15
IPC codes: A61M1, C07K14, G01N33