HEME CONCENTRATE AND METHOD FOR THE PREPARATION THEREOF

Abstract: A heme concentrate containing 15 to 55 percent heme in readily absorbable form and useful as a pharmaceutical against anemia and as an iron-enriching agent in foodstuffs, and a method for the preparation of said heme concentrate of a mixture of heme and blood substance obtained when cleaving hemoglobin, for example, from whole blood or red blood cells, by treating said mixture with a dehydrating agent, preferably a lower alcohol or a mixture of alcohols, either at a pH of at least 8.0 or in the presence of a substance promoting the separation, preferably imidazole or an imidazole derivative, at a pH of at least 6.0, preferably 6.5 to 8.5, whereafter the solid blood substance is separated and the heme concentrate is recovered from the remaining solution; the blood substance simultaneously recovered, mainly globin, is intended for purposes of foodstuffs and fodder.

Description: Heme concentrate and method for the preparation thereof . Slaughtering blood contains valuable components, namely protein having a high nutritive value and functional properties, which make it useful as texturing agent in foodstuffs , and further heme which is a sought-after iron- enriching agent. The hemoglobin, which contains the main part of the protein in the blood, has a taste of iron because of the heme. It is therefore of interest to develop a method for cleaving the hemoglobin into an iron-free globin and a heme concentrate, and in such a way that the functional proper¬ ties of the protein and the heme are preserved so as to give these products a high value. As is well-known, protein is easily denatured and therewith looses its functional properties, for example, when treated at a high pH (over 9 to 10) . The denaturation can be counteracted by operating at a low temperature, less than 0 O C. in order that heme may be useful as a pharmaceutical, i.e. in order that the iron be present in a readily ab- sorbable form, the heme must be present in native form. In hemoglobin, the heme is present in native form out of which the iron, as krown, is readily absorbed. out of conventional iron preparations, such as iron sulfate, only a fraction of the iron is absorbed because certain substances present in the food (e.g., phytates) prevent the absorption. This drawback is not present in iron based on heme. Heme has a pronounced tendency to polymerize and to form difficultly soluble aggregates out of which the iron cannot be absorbed. Known methods for cleaving the hemoglobin into heme and globin and for separating the heme by extraction with acetone or methyl ethyl ketone result in a polymerized heme. The present invention relates to a method for recovering a heme concentrate from a mixture of heme and blood substance obtained when cleaving hemoglobin. Heme, iron heme, is composed of iron and proto- porphyrin (see, for example, Textbook of Biochemistry, West & Todd, MacMillan Co., 1961, p. 533) and constitutes the prosthetic group in hemoglobin and amounts to 3.8 per cent by weight based on the hemoglobin. As starting material, a heme product is preferably used which -is obtained by cleaving hemoglobin into heme and globin. The cleavage can be carried out in different ways , for example, by treating hemoglobin in an acid or alkaline milieu. The " raw material can comprise, for example, whole blood, red blood cells, or hemoglobin in another milieu. A particularly suitable starting material is the heme product obtained according to the method described in the Swedish Patent No. 7407882-5 and in the Swedish Patent Application No. 7513987-3. According to this method the hemoglobin is cleaved in an aqueous ethanol solution having an ethanol content of at least 40 per cent by volume, at a pH of less than 4.5. The precipitate of heme product formed thereby is separated. The remaining globin solution is practically free of heme. This known product contains about 10 per cent by weight of heme while the rest consists of other blood substance, most likely mainly of protein. Moreover, this heme product, in all probability, contains blood substance such as lipoproteins, lipides, phosphatidyl compounds and cholesterol. According to the new method, a heme concentrate is obtained having a heme content of about 40 per cent by weight. The composition is not known. This heme concentrat is intended for use as a pharmaceutical against anemia and and as an iron-enriching agent in foodstuffs. The blood protein, mainly globin, which is simul- taneously recovered, is intended for purposes of food¬ stuffs and fodder. The method is technically and economically feasibly on industrial scale. According to the method according to the invention, said heme raw material is treated with a dehydrating agent, preferably a lower alcohol or a mixture of alcohols and/or ketones and esters, said treatment taking place either a) at a pH of at least 8.0, preferably 9.0 to 10.5, or b) in the presence of a compound of the formula: lb Ic where the substituents can have the following meanings: -R (R is a saturated or unsaturated alkyl or aryl or CH_) -H, -R (R) , -COOH, -R(COOH) , -COOR, — R(COOR) , n' - ' . -OH, -ROH, -R(OH) , -R(OH) R, -OR, -ROR, -R(OR) , n -NH 2 , -R(NH , ~ NR 2' -*^ JR 2' ~ N 2 R ' -CN, -R(CN) n , -Cl, -R(Cl) n , -F, -R( ) n , -3r, -R(Br) n , -J, -R(J) n _N0 2' ~ R(N0 2 ) n' -COR, -R(COR) , -R(COCOOH) , -R(COCOOR) , -OCOR, n n n -SH, -R(SH) H, -SO H, -RNHCOOH, -RNHCOOR, -R(NH) R(OH) , -R(NHCOR) COOH, n n n -R(NHCOR) COOR, -NHCOOH, -NHCOOR, -R(NHCOOH) , -R(NHCOOR) n , -R(NH„) (COOH) , 2 n n -R(NH 2 ) (COOR) n , -R(COOH) 1ΪHCOOH, -R (COOR) n NHCOOR, -RN(R)ROH, -CONH_, -R(CONH„) , -CONHl.H.. , -R(CONHNH„) , z z n z z Ϊ -NHCONH„, -R(NHCONH„) , 2 2 n t. Όx-mb VA . t?- -NHCOR, -RNHCOR, -CONHR, -RCONHR, -CON- , -R(CON3_)n' -RCOOHNHCORNH 2' -S0 2 NHR, -S0 2 RNHCOOR, -CONHNHSO R, -RSO R, -NHN0 2 , -RON0 2 , -RC1C00H,-RC1C00R, -RFCOOH, -RFCOOR, -RBrCOOH, -RBrCOOR, -RJCOOH, -RJCOOR, and where, in addition, X. and X', and/or X_ and X_ * , can be substituted (replaced) by =0, =NH, =S, =CHR(N0 2 ) , =N(H«PO.) or =CHR, and for the imidazoline compound lb, in addition, X. and X ' and/or X_ and X_- ' . and for the imid¬ azoline compound Ic, moreover, X~ and X ? ' , X, and X. ' , and/or X,- and X- ' , also can be substituted by =0, =NH, =S, =CHR(N0 2 ) , =N(H 2 P0.) and =CHR, in which substituent formulas n is a number from 1 to 5, or any other similar compound, such as: Anserine NHCOCH 2 CH 2 NH 2 Carnosine SH Ergotioneine 3 Histamine Imidazolyl-CH 2 CH 2 NH 2 Histidole Imidazolyl-CH 2 CH 2 OH Dimethylhistamine Histidine Pilocarpine CH CH, Pilosinine SH I -C. Thiolhistidine - HN ^N NH i HC ■ C-CH CHCOOH s Hercynine Imidazolyl glycine xmidazolyl-CH (NH_) COOH7 Imidazolyl lactic acid (imidazolyl-CH 2 CHOHCOOH) Imidazolyl propionic acid (imidazolyl-CH 2 CH 2 COOH) ϋrocanic acid (imidazolyl-CH=CHCOOH) Histamine-tyramine (HOC 6,H4.CH2_CH 0 NHCHZCH2_-imidazolvl) Imidazolyl acidic acid (imidazolyl-CH_COOH) Sulfaimidazoles Vitimidazole / _T ; ' ■' ____ SH I C Thiolhistamine HN N HC C-CH 2 CH NH at a pH of at least 6.0, preferably 6.5 to 8.5 whereafter the undissolved blood substance is separated, e.g., by centrifugation or filtration and the heme concentrate is precipitated from the remaining solution by lowering pH to a value less than 6.5, and is recovered. It is previosly known to cleave hemoglobin into hem and globin in an alkaline milieu while extracting the heme for example, in acetone or methyl ethyl ketone to separate it in this way from the globin. This extraction is carried out in 100 % acetone to give a pure heme without any other blood substance. However, this heme has proved to poly¬ merize and has a poor solubility (see Figure 2) . From the Swedish Patent Publication 76-00020-7 it • is known to prepare an iron preparation based on hemoglobi by linking iron porphyrin to hemoglobin or globin at a pH of 8 to 9. According to the new method, the heme concentrate is separated from any other blood substance, i.e. mainly protein, so that the heme concentrate is converted into the dissolved form while the remaining substance is presen in ' undissolved form. Suitable extraction agents are ethanol, methanol, propanol, isopropanol, butanol, isobutanol, ethylene glyco and glycerol. Ethanol is preferred because it is not toxic. The content of extraction agent must be over 40 per cent by weight, preferably over 60 per cent by weight. The pH value must, in the presence of a compound of the formula I, be at least 6.0, preferably 6.5 to 8.5. Otherwise, the pH value must be at least 8.0, preferably 9.0 to- 10.5 In order to prevent denaturation of the protein the temperature is maintained low, preferably less than -5°C. The. temperatur does not affect the reaction, only the degree of denaturation of the protein. In order to prevent dissolution of blood substance from the heme/blood substance mixture simultaneously with the heme concentrate in the extraction solution, the blood substance, mainly globin, can be precipitated before the extraction. The precipitation can be effected, for example, by adjusting the pH for the mixture to a value of 7 to 8, the isoelectric pH range for globin, and/or by adding various salts. Suitable salts are, for example, Na, K, NH. chloride, sulphate, citrate, phosphate, tartrate. The addition of salt has the conventional salting-out effect on proteins but has no special effect on the dissol¬ ution of the heme concentrate. The addition of salt mainly comes into question when the extraction is carried out on a heme mixture from which the globin portion has not been previously separated. in addition, other agents promoting the separation can be added, such as amino acid, e.g., glycine, glutamine, or lysine. Glycine has the effect of increasing the yield of heme concentrate up to about 99 per cent, see Example 8, when the other conditions at the same time are ideal. Without the addition of glycine, the yield remains at about 93 to 95 per cent. The undissolved substance is separated, for example, by centrifugation or filtration. The heme concentrate is precipitated out of the remaining solution, i.e. the supernatant or the filtrate, by lowering the pH value to less than 6.5, preferably 4.0 to 6.0. A black, flaky precipitate is then formed. The precipitation will be more quantitative when the pH value i s less than 5. The precipitation is rather undependent on the temperature, but in order to obtain a more quantitative precipitation and in order to subject the concentrate to a careful treatment, the precipitation is preferably carried out at a temperature of less than 0 C. The pH is lowered, for example, with 0.1 M HC1. The precipitate is separated, for example, by centrifugation or filtration, and is washed free of ethanol, salt and imidazole with ice water. The precipitate is dissolved in water by raising the pH, for example, to 8, and the solution can be freezed and freeze dried. The yield of heme out of the extraction solutions amounts to about 90 per cent. Because the separation of heme concentrate can be carried out at a pH of less than 10.5 and at. a temperature lower than 0 C, the protein is recovered in a non-denature or an only partially denatured form whereby the functional properties thereof are preserved. In connection with food¬ stuffs, it is important to aim at proteins with texturing properties. The protein will be particularly satisfactory when recovered at a pH lower than about 9. The heme concentrate obtained in the method accordi to the invention usually has a heme content of about 35 to 40 per cent; in special cases, products having a lower (15 per cent) and also a higher (up to 55 per cent) heme content can be obtained. The heme concentrate possibly is a new chemical compound consisting of heme and blood substance. The compo sition of the portion herein referred to as "blood substan is not known. The heme concentrate cannot be physically split " into its components, e.g., by gel filtration. Obvi¬ ously, the compound is formed under the conditions pre¬ vailing when the heme is cleaved from the hemoglobin. In an aqueous ethanol solution having an ethanol content of more than 40 per cent by volume and pH values higher than 6.0, this compound is freed from other blood substanc Since the solubility is good, it is easily separated from other blood substance which is not soluble under these conditions The presence of a compound of the formula I, such as imidazole, imidazoline, or imidazolidine, or a deriva¬ tive thereof, facilitates the separation of the heme concentrate from adhering blood substance so that quan- titative separation can be effected already at a pH value of 7. The good solubility of the heme concentrate indicates that the heme is present in native form, not polymerized. Therefore, it is possible for the iron to be absorbed out of the heme concentrate, and it can then be used as an iron-enriching agent in foodstuffs and as a pharma¬ ceutical against anemia. The solubility of the heme concentrate Figure 2 illustrates the solubility of the heme concentrate at different pH values . The heme concentrate 1 is representative of the product prepared according to the invention. When treating the concentrate with a proteolytic enzyme, e.g., with trypsin, the concentrate is cleaved into smaller units and the solubility is somewhat improved; the heme concentrate 2 elucidates this. For the sake of comparison, the solubility curve for hemin has been plotted (prepared by Nishida, Porphyrins and Metalloporphyrins, Kevin M. Smith, 1975, page 809) . AS mentioned earlier, the heme preparation must be soluble at a pH value which is lower than or equal to the pH value, 7.5 to 8.2, which prevails in the intestine where the absorption of iron takes place. The absorption of iron out of the heme concentrate T e absorption of iron was determined by means of clinical experiments. To ten test persons was served an aqueous solution of heme concentrate marked with the isotope 55Fe. As a substance of comparison was used blood marked with the isotope Fe. The absorbed iron quantity was determined by means of radioactive measurement. The results are presented in Table I. The results show that the absorption of iron out of the heme concentrate was approximately of the. same order, often even somewhat higher, than out of blood. This can be interpreted so that the heme concentrate exists in native form, which is the prerequisite for an absorption o iron. Table I The absorption of iron -.out of heme concentrate Test person Age ight Hemo¬ Hct Absorption (%) Absorbed quantity ( g) male/female (years) (cm) kg) globin (%) Blood Heme Blood value Heme con¬ con¬ centrate (g %) centrate f 26 162 55 13,2 40 15,5 12,3 0,78 0,62 23,2 m 33 179 69 13,8 45 8,3 7,7 0,41 0,39 30,8 f 37 164 49 14,2 44 8,1 8,6 0,41 0,43 32,7 f 30 165 58 13,2 42 5,3 5,6 0,27 0,28 32,1 m 36 170 69 15,3 48 7,0 6,8 0,35 0,34 46,9 m 33 181 80 15,6 49 8,5 8,7 0,43 0,44 73,0 m 24 183 76 14,9 48 13,3 13,5 0,67 0,68 92,4 m 34 180 86 14,3 42 6,4 10,9 0,32 0,55 68,0 m 26 184 90 14,7 45 11,1 16,3 0,56 0,82 ' 70,6 m 30 176 73 14,0 44 6,6 10,8 0,33 0,54 37,8 • Mean value: . 9,0 10,1 0,45 0,51 50,8 SEM: 1,04 1,04 0,05 0,05 7,40 Example 1 A mixture of 4 ml of 96 %-by-volume ethanol and 0.8 ml of 1 M HC1 at a temperature of -8°C was added drop- wise, while stirring and cooling, to 2.2 g of a mixture of blood cells at a temperature of -5°C. After the additio the pH value was 3.1 and the temperature -5 C. * 3 ml of ice water and 1.8 ml of 0.5 M NaOH in 70 %-by-volume ethanol was added while stirring and cooling; the temperature of the mixture was -5°C and the pH 7.5. After 5 minutes, 10 m of 96 %-by-volume ethanol, 10 ml of 70 %-by-volume ethanol 1 ml of 1 M glycine in an aqueous solution and 2.2 ml of 0.5 M NaOH were added to the mixture while stirring and cooling. The mixture now contained 67 %-by-volume ethanol, the pH was 10.3 and the temperature -5°C. The mixture was centrifuged at 3000 x g for 5 minutes, whereby 38 ml of a red supernatant and a brownish red paste were obtained. The supernatant contained 0.18 mg of heme/ml (61.5 % of th heme content of the starting material) . The paste was mixe in 10 ml of 70 %-by-volume ethanol at a temperature of -5° the pH was adjusted to 10.3, and the mixture was centrifug as above. The supernatant contained 0.13 mg of heme/ml (11.7 % of the heme content of the starting material) . The paste was treated for a third time in the same manner as above, whereby 0.06 mg of heme/ml (5.4 % of the heme conte of the starting material) was obtained. The total yield of heme concentrate was 78.6 %. The paste was brown and weighed in dry condition 0.27 g (protein yield 79.5 %) . The above mentioned mixture of blood cells can be - obtained in the following manner. Slaughtering blood, with sodium citrate as anti¬ coagulant, was separated by centrifugation, whereby plasma and a broth of red blood cells were obtained. Sodium chlor ide and ethanol were added to the broth of blood cells so that 2.2 g of the mixture of blood cells contained 0.34 g protein, 0.16 g sodium chloride, 0.26 g of 100 % ethanol. and 1.44 g water, and soluble substances present in the blood. The protein, mainly hemoglobin, was determined by means of precipitation with ethanol. 2.2 g of the mixture of blood cells contained 11.1 mg hemin, determined accord¬ ing to the pyridine method. Example 2 In the same way as in Example 1, the pH was lowered to 3.1 and thereafter adjusted to 7.5. The mixture was extracted in the same way as in Example 1 but, in addition, by adding 1 ml of 2 M imidazole in 70 %-by-volume ethanol. The pH was adjusted to 8.4. The supernatants contained 79 %, 12 % and 4 %, respectively, of the heme content of the starting material. The total yield of heme concentrate was 95 %. The paste was light brown and represented 85 % of the protein content of the starting material. Example 3 A mixture of 2 ml water and 0.7 ml of 1 M HC1 at a temperature of +1 C was added dropv/ise, while stirring and cooling, to 2.2 g of a mixture of blood cells at a tempera¬ ture of -8 C. After the addition, the pH was 3.2 and the • temperature -5 C. 4 ml of 96 %-by-volume ethanol, 10 ml of 70 %-by-volume ethanol, 1 ml of 1 M glycine in an aqueous solution, 1 ml of 2 M imidazole in 70 %-by-volume ethanol and 1.5 ml of 0.5 M NaOH in 70 %-by-volume ethanol were added, while stirring and cooling; the temperature of the mixture was -5°C and pH 8.0, the ethanol content was about 56 %-by-volume. The mixture was centrifuged after 5 minutes as in Example 1 and yielded a supernatant con¬ taining 59 % of the heme content of the starting material and a brown paste. The paste was extracted two times with 10 ml of 70 %-by-volume ethanol, each time at a pH of 8.0, and yielded supernatants containing 18 % and 8 %, respect¬ ively, of the heme content of the starting material. The total yield of heme concentrate was 85 %. The paste was brown and represented 72 % of the protein content of the starting material. Example 4 A mixture of 3 ml of 96 %-by-volume ethanol and 0.35 ml of 0.5 M NaOH in 70 %-by-volume ethanol at a tempera- ture of —10 C was added dropwise, while stirring and cooli to 2.2 * g of a mixture of blood cells at a temperature of -10 C. After the addition, the pH was 10.2 and the tempera ture -10°C. A mixture of 2 ml of ice water, 3 ml of 50 %-b volume ethanol and 0.15 ml of 1 M HCl, at a temperature of -10°C were added. The pH of the mixture was now 7.5 and th temperature -10 C. A mixture of 5 ml of 96 %-by-volume ethanol, 5 ml of 70 %-by-volume ethanol and 2 ml of 0.5 M NaOH in 70 %-by-volume ethanol, at a temperature of -10°C were added. The pH of the mixture was 10.3, the temperatur -10°C, and the ethanol content about 64 %-by-volume. The mixture was centrifuged after 5 minutes as in Example 1 an yielded a supernatant containing 46 % of the heme content of the starting material and a dark brown paste. The paste was extracted twice with 10 ml of 65 %-by-volume ethanol, each time at a pH of 10.3 and a temperature of -10°C, and yielded supernatants containing 14 % and 7 %, .respectively, of the heme content of the .starting material. The total yield 'of heme concentrate was 67 %. The paste was brown an represented 71 % of the protein content of the starting material. Example 5 In the same way as in Example 4, the pH of the mixture of blood cells was raised to 10.2, the temperature 7as maintained at 0 C. A mixture of 1.5 ml of ice water, 3 ml of 50 %—by-volume ethanol, 1 ml of 2 M imidazole in 70 %-by-volume ethanol and 0.2 ml of 1 M HCl, at a tempera¬ ture of 0 C was added. The pH of the mixture was 8.2 and th temperature 0 C. Hereafter a mixture of 5 ml of 96 %-by- volume ethanol and 10 ml of 70 %-by-volume ethanol, at a temperature of 0 C were added. The pH of the mixture was 8.2, the temperature 0 C, and the ethanol content 66 % by volume. The mixture was centrifuged after 5 minutes as in Example 1 and yielded a supernatant containing 52 % of the heme content of the starting material and a dark brown paste. The paste was extracted twice with 10 ml of 65 %-by- volune ethanol and 0.2 ml of 2 M imidazole in 70 %-by-volu ethanol, each time at a pH of 8.2 and a temperature of 0°C, and yielded supernatants containing 28 % and 12 %, respect¬ ively, of the heme content of the starting material. The total yield of heme concentrate was 92 % . The paste was brown and represented 85 % of the protein content of the starting material. Example 6 A mixture of 1 ml water and 0.8 ml of 0.5 M NaOH at a temperature of +10 C was added dropwise, while stirring, to 2.2 g of a mixture of blood cells at a temperature of +10 C. After the addition, the pH was 11.0 and the tempera¬ ture +10 C. 4 ml of 96 %-by-volume of ethanol, 1 ml of 2 M imidazole in 70 %-by-volume ethanol, 1 ml of 30 % sodium citrate in an aqueous solution and 0.5 ml of 1 M HCl were added. The pH of the mixture was 7.5 and the temperature +10 C. Hereafter 3 ml of 96 %-by-volume ethanol and 10 ml of 70 %-by-volume ethanol were added. The pH of the misture was 7.5, the temperature +10 C and the ethanol content about 64 %-by-volume. The mixture was centrifuged after 5 minutes as in Example 1 and yielded a supernatant con¬ taining 53 % of the heme content of the starting material and a dark brown paste. The paste was extracted twice with 10 ml of 65 %-by-volume ethanol and 0.1 ml of 2 M imidazole in 70 %-by-volume ethanol, each time at a pH of 7.5 and a temperature of +10 C, and yielded supernatants containing 26 % and 13 %, respectively, of the heme content of the starting material. The total yield of heme concentrate was 92 %. The paste was brown and represented 74 % of the protein content of the starting material. Example 7 A mixture of 5.5 ml of 96 %-by-volume ethanol and 1.0 ml of 1 M HCl, at a temperature of -18 C was added dropwise, while stirring and cooling, to a mixture of 2.2 g of a mixture of blood cells, 0.1 ml of 1 M glycine in an aqueous solution, 0.1 ml of 2 M imidazole in 70 %-by-volume ethanol and 2 ml of 50 %-by-volume*ethanol, at a temperature of -18 C. After the addition, the pH was 3.0 and the temperature -18°C. 3 ml of 40-% ammonium sulphate in an aqueous solution was added while stirring and cooling. After 5 minutes, a mixture of 10 ml of 96 %-by-volume of ethanol, 20 ml of 70 %-by-volume ethanol and 4 ml of 0.5 M NaOH in 70 %-by-volume ethanol, at a temperature of -18 C was added while stirring and cooling. After the addition, the pH was 7.5 and the temperature -18 C, the ethanol content was about 69 % by volume. The mixture was centri¬ fuged as in Example 1. The paste was extracted 2 times wit 20 ml of 70 %-by-volume ethanol, 0.1 ml of 1 M glycine in an aqueous solution and 0.1 ml of 2 M imidazole in 70-%-by volume ethanol, each time at a pH of 7.5 and a temperature of -18 C. The supernatant contained 69 %, 14 % and 5 %, respectively, of the heme content of the starting material The total yield of heme concentrate was 88 %. The paste was light brown and represented 81 % of the protein conten of the starting material. Example 8 A mixture of 8 ml of 96 %-by-volume ethanol and * 0.8 ml of 1 M HCl at a temperature of -5°C was added drop¬ wise, while stirring and cooling, to 2.2 g of a mixture of blood cells at a temperature of -5 C. After the additio the pH was 3.1 and the temperature -5°C. 0.8 ml of 0.2 M NaOH in 50 %-by-volume ethanol and 10 ml of 50 %-by-voluιr.e ethanol were added, the pH was 3.5 and the temperature -5 C. The mixture was centrifuged at 9000 x g for 5 minute whereby a a yellow supernatant and a black paste were obtained. Out of the supernatant, after the addition of 8 ml of water, at a pH of 7.5, a grey protein precipitate containing 0.21 g of dry substance was precipitated. The black heme paste was mixed with a mixture of 30 ml of 70 %-by-volume ethanol, 0.5 ml of 1 M glycine in an aqueou solution, 0.5 ml of 2 M imidazole in 70 %-by-volume ethano and 2.5 ml of 0.5 M NaOH in 70 %-by-volume ethanol. The pH was 8.6, the temperature -5°C, and the ethanol content about 70 % by volume. The mixture was centrifuged at 3000 x g for 5 minutes and yielded a supernatant containin 72 % of the heme content of the starting material and a brown paste. The paste was- extracted twice with 30 ml of 70 %-by- volume ethanol and 0.1 ml of 2 M imidazole in 70 %-by-volume ethanol, each time at a pH of 8.6 and a temperature of -5 C, and yielded supernatants containing 19 % and 8 %, respect¬ ively, of the heme content of the starting material. The total yield of heme concentrate was 99 %. The paste was greyish brown and weighed as dry 0.1 g. The total protein yield was 91 %. Example 9 a) In the same way as in Example 8, the black heme paste was separated starting from 2.2 g of a mixture of blood cells. The paste was mixed with 30 ml of 50 %-by- volume ethanol and pH was adjusted to 8.5 with 0.5 M NaOH in 70 %-by-volume ethanol, at a temperature of -1 C. The ethanol content was about 50 %-by-volume. The mixture was centrifuged as in Example 8 and yielded a supernatant containing 33 % of the heme content of the starting material .and a dark brown paste. The paste was extracted 3 times with 30 ml of 50 %-by-volume ethanol, each time at a pH of 9.0 and a temperature of -1 C, and yielded supernatants containing 24 %, 13 % and 9 %, respectively, of the heme content of the starting material. The total yield of heme concentrate was 79 % . The paste was brown and weighed as dry 0.1 g. The total protein yield was 91 %. b) In the same way as in Example 8, the black heme paste was separated starting from 2.2 g of a mixture of ' blood cells. The paste was mixed with 30 ml of 40 %-by- volume ethanol and the pH was adjusted to 10.7 with 0.5 M NaOH in 70 %-by-volume ethanol. The temperature was -4°C and the ethanol content about 40 %-by-volume. The mixture was centrifuged as in Example 8 and yielded a supernatant containing 71 % of the heme content of the starting ma¬ terial and a brown paste. The paste was extracted twice with 30 ml of 40 %-by-volume ethanol, each time at a pH of 10.7 and a temperature of -4°C, and yielded supernatants containing 7.2 % and 4.8 %, respectively, of the heme content of the starting material. The total yield of heme concentrate was 83 %. The paste was • light brown and weighe as dry 0.08 g. The total protein yield was 85 %. Example 10 In the same way as in Example 8, the black heme paste was separated starting from 2.2 g of a mixture of blood cells . The paste was mized with a mixture of 20 ml of 60 %-by-volume ethanol, 0.2 ml of 2 M imidazole in 70 %-by-volume ethanol and 1 ml of 40 %-by-weight of ammonium sulphate in an aqueous solution. The pH was ad¬ justed to .6.8 with 0.5 M NaOH in 70 %-by-volume ethanol. The temperature was +25 C and the ethanol content about 60 %-by-volume. The mixture was centrifuged as in Example and yielded a supernatant containing* 63 % of the heme content of the starting material and a brownish red paste. The paste was extracted twice with 60 %-by-volume ethanol, imidazole and ammonium sulphate in quantities as above, each time at a pH of 6.8 and a temperature of +25 C, and yielded supernatants containing 14 % and 8 %, respectively of the heme content of the starting material. The total yield of heme concentrate was 85 %. The paste was brown an weighed as dry 0.07 g. The total protein yield was 83 %. Example 11 In the same way as in Example 8, the black heme pas was separated starting from 2.2 g of a mixture of blood cells. The paste was mixed with a mixture of 30 ml of 96 %-by-volume ethanol and 0.2 ml of 1 M glycine in an aqueous solution. The pH was adjusted to 10.4 with 0.5 M NaOH in 70 %-by-volume ethanol. The temperature was -20 C, the ethanol content about 90 %-by-volume. The mixture was centrifuged as in Example 8 and yielded a supernatant containing 61 % of the heme content of the starting ma¬ terial and a dark brown paste. The paste was extracted twice with 90 %-by-volume ethanol and glycine in quantitie as above, each time at a pH of 10.4 and a temperature of -20 C, and yielded supernatants containing 20 % and 9 %, respectively, of the heme content of the starting material The total yield of heme concentrate was 90 %. The paste was light brown and weighed as dry 0.1 g. The total protein yield was 91 %. Example 12 In the same way as in Example 8, the black heme paste was separated starting from 2.2 g of a mixture of blood cells. The paste was mixed with a mixture of 30 ml of 40 %-by-volume ethanol and 0.2 ml of 2 M imidazole in 70 %-by-volume ethanol. The pH was adjusted to 7.0 with 0.5 M NaOH in 70 %-by-volume ethanol. The temperature was -5 C, the ethanol content about 42 %-by-volume. The mixture was centrifuged as in Example 8 and yielded a supernatant containing 58 % of the heme content of the starting material and a dark red paste. The paste was extracted 2 times with 70 %-by-volume ethanol and imidazole in quantities as above, each time at a pH of 7 and a temperature of -5 C, and yielded supernatants containing 26 % and 5 %, respectively, of the heme content of the starting material. The total yield of' heme concentrate was 89 %. -The paste was light brown and weighed as dry 0.1 g. The total protein yield was 91 %. Example 13 In the same way as in Example 8, the black heme paste was separated starting from 2.2 g of a mixture of blood cells. The paste was mixed with a mixture of 20 ml of 70 %-by-volume methanol and 0.2 ml of 2 M imidazole in 70 %-by-volume ethanol. The pH was adjusted to 8.5 with 0.5 M NaOH in 70 %-by-volume ethanol. The temperature was -5°C, the methanol content about 60 %-by-volume. The mixture was centrifuged as in Example 8 and yielded a supernatant containing 67 % of the heme content of the starting material and a brownish red paste. The paste was extracted twice with 70 %-by-volume methanol and imidazole in quantities as above, each time at a pH of 8.5 and a temperature of -5 C, and yielded supernatants containing 18 % and 7 % , respect- ively, of the heme content of the starting material. The total yield of heme concentrate was 92 %. The paste was light brown and weighed as dry 0.1 g. The total protein yield was 91 % . Example 14 In the same way as in Example 8, the black heme paste was separated starting from 2.2 g of a mixture of blood cells. The paste was mixed with a mixture of 20 ml of 70 %-by-volume propanol and 0.2 ml of 2 M imidazole in 70 %-by-volume ethanol. The pH was adjusted to 8.0 with 0.5 M NaOH in 70 %-by-volume ethanol. The temperature was -5 C, the propanol content , about 60 %-by-volume- The mixt was centrifuged as in Example 8 and yielded a supernatant containing 66 % of the heme content of the starting materi and a brownish red paste. The paste was . extracted 2 times with- 70 %-by-volume propanol and imidazole in quantities a above, each time at a pH of 8.0 and a temperature of -5 C, and yielded supernatants containing 16 % and 9 % , respect- ively, of the heme content of the starting material. The total yield of heme concentrate was 91 %. The paste was light brown and weighed as dry 0.1 g. The total protein yield was 91 %. * Example 1 ' 5 n the same way as in Example 8, the black heme pas was separated starting from 2.2 g of a mixture of blood cells. The paste was mixed with a mixture of 20 ml of 70 %-by-volume isopropanol, 0,2 ml of 1 M glycine and 0.2 of 2 M imidazole in 70 %-by-volume ethanol. The pH was adjusted to 7.5 with 0.5 M NaOH in 70 %-by-volume ethanol. The temperature was -10 C, the isopropanol content about 60 %-by-volume. The mixture was centrifuged as in Example and yielded a supernatant containing 69 % of the heme con¬ tent of the starting material and a brownish red paste. Th paste was extracted twice with 70 %-by-volume isopropanol, glycine and imidazole in quantities as above, each time at a pH of 7.5 and a temperature of -10°C, and yielded super¬ natants containing 17 % and 9 %, respectively, of the heme content of the starting material. The total yield of heme concentrate was 95 %. The paste was light brown and weighe as dry 0.1 g. The total protein yield was 91 %. Ot--? V _ Ir_ : Example 16 In the same way as in Example 8, the black heme paste was separated starting from 2.2 g of a mixture of blood cells. The paste was mixed with a mixture of 20 ml of 70 %-by-volume ethanol, 7 ml of 70 %-by-volume glycerine and 0.2 ml of 2 M imidazole in 70 %-by-volume ethanol. The pH was adjusted to 8.0 with 0.5 M NaOH in 70 %-by-volume ethanol. The temperature was -5 C, the ethanol content about 52 %-by-volume and the glyserine content about 17 %- by-volume. The mixture was centrifuged as in Example 8 and yielded a supernatant containing 64 % of the heme content of the starting material and a brownish red paste. The paste was extracted twice with 70 %-by-volume ethanol, 70 %-by- volume glycerine and imidazole in quantities as above, each time at a pH of 8.0 and a temperature of -5 C, and yielded supernatants containing 14 % and 7 %, respectively, of the heme content of the starting material. The total yield of heme concentrate was 85 %. The paste was brown and weighed as dry after washing with water 0.1 g. The total protein yield was 91 %. In the same way as above, an extraction was carried out with 70 %-by-volume ethylene glycol instead of glycer¬ ine. The result was the same as above. Example 17 A mixture of 6 ml of 96 %-by-volume ethanol and 1 ml of 1 M HCl at a temperature of -8 C was added dropwise, while stirring and cooling, to 2.7 g of whole blood at a temperature of +0.5 C. After the addition, the pH was 3.0 and the temperature -5°C. Hereafter 2 ml of 40 % ammonium sulphate in an aqueous solution and a mixture of 20 ml of 70 %-by-volume ethanol, 0.2 ml of 1 M glycine in an aqueous solution and 0.2 ml of 2 M imidazole in 70 %-by-volume ethanol at a temperature of -8°C were added while stirring and cooling. The pH was adjusted to 8.0 with 0.5 M NaOH in 70 %-by-volume ethanol. The temperature was -8°C and the ethanol content about 65 %-by-volume. The mixture was cen¬ trifuged as in Example 8 and yielded a supernatant containing 66 % of the heme conten -of the starting material and a brownish red paste. The paste was extracted twice with 70 %-by-volume ethanol, ammonium sulphate and imidazole in quantities as above, each time at a pH of 8.0 and a temper ture of -8 C, and yielded supernatants containing 16 % and 7 %, respectively, of the heme content of the starting material. The total yield of heme concentrate was 89 %.The was greyish brown after washing with 50 %-by-volume ethano and represented 85 % of the total protein content. Example 18 An extraction of heme paste starting from 22 g of a mixture of blood cells yielded at a pH of 10.4, a temper ture of -5 C and 70 %-by-volume ethanol the first extracti solution of 199 ml with the heme content of 0.38 mg heme/m representing 68.1 % of the heme content of the starting material, the second extraction solution of 121 ml with th heme content of 0.17 mg heme/ml representing 18.6 % of the heme content and the third extraction solution of 118 ml,, with the heme -content of 0.08 mg heme/ml representing 8.5 of the heme content.. The extraction solutions were combine and acidated to a pH of 5.1 with 87 ml of 0.1 M HCl, at the temperature of -5 C. The black flaky precipitate forme hereby was separated in a centrifuge at 3000 x g and weigh 4.5 g. The paste was washed 3 times in 15 ml of ice water each time, suspended in 15 ml of ice water, the pH was adjusted to 8.2 with 3 ml of 0.2 M NaOH, freezed and freez dried. Hereby 265 mg of a black powder with a heme content of 36 % was obtained. The total yield of heme concentrate was 86 %. Analysis: C 46.17 * %, H 5.30 %, Cl 10.53 %, P 2.8 Fe 3.57 %, N 9.6 %, K 3.5 % and Na 4.1 %. Example 19 An extraction of heme paste starting from 50 g of a mixture of blood cells yielded at a pH o ' f 8.2, a tempera ture of +1 C and with 60 %-by-volume ethanol and 4 ml of 2 imidazole in 70 %-by-volume ethanol the first extraction solution of 494 ml with the heme content of 0.32 heme/ml representing 62.8 % of the heme content of the starting material, the second extraction solution with corresponding data 467 ml, 0.11 mg heme/ml, 20.4 % and the third extrac¬ tion solution with corresponding data 479 ml, 0.06 mg heme/ml, 11.4 %. The extraction solutions were combined and acidated to a pH of 4.0 with 240 ml of 0.1 M HCl, at the temperature of +1 C. The precipitate was separated as above and weighed 8.5 g. The paste was washed 3 times in 25 ml of ice water each time, suspended in 25 ml of ice water, the pH was ad¬ justed to 8.5, and the suspension was freezed and freeze dried. Hereby 510 mg of a black powder with a heme content of 42 % was obtained. The total yield of heme concentrate was 85 %. Example 20 In the same way as in Example 18, three extraction solutions were obtained. The third extraction solution was acidated separately to a pH of 5.1 and yielded a smaller amount of precipitate which, after freeze drying, yielded a heme concentrate with a heme content of 17.7 %. Analysis: C 29.42 %, H 3.52 %, Cl 23.77 %, P 1.2 %, Fe 2.28 %, N 8.8 %, ' K 13.4 % and Na 15.7 %. Example 21 In the same way as in Example 19 , one extraction solution was obtained. The extraction solution was acedu- lated separately to a pH 4.0 and yielded a black precipitate which was separated, washed and freeze dried in the same way as in Example 19. The heme concentrate thus obtained had a heme content of 55 %.

Claims: Claims : 1. A method for the preparation of a heme concentra of a mixture of heme and blood substance obtained when cleaving hemoglobin, c h a r a c t e r i z e d in that said mixture of heme and blood substance is treated with a dehydrating agent, preferably a lower alcohol or a mixtu of alcohols and, in addition, possibly ketones and esters, said treatment taking place either a) at a pH of at least 8.0, preferably 9.0 to 10.5, or b) in the presence of an agent promoting the separ¬ ation, preferably imidazole or an imidazole derivative, at a pH of at least 6.0, preferably 6.5 to 8.5, and wherein agents stabilizing the precipitation of protei possibly added, whereafter the solid blood substance is separated, and the heme concentrate is recovered from the remaining solution. 2. A method as claimed in claim 1, c h a r ' a c - t e r i z e d in that the mixture of heme and blood sub¬ stance comprises a heme paste obtained by cleaving hemo¬ globin substance and by separating the globin solution in a manner known per se. 3. A method as claimed in claim 1 and 2, c h a r a c t e r i z e d in that agents stabilizing the precipitation of proteins, such as one or more salts, e.g. sodium, potassium or ammonium chloride, sulphate, phos¬ phate, citrate or tartrate, are added. 4. A method as claimed in claims 1 to 3, c h a r a c t e r i z e d in that as agents promoting th separation are added a ino acids, preferably glycine, glutamine or lysine. 5. A method as claimed in claim 1, wherein a mix¬ ture of heme and blood substance obtained when cleaving hemoglobin is used as starting material, c h a r a c ¬ t e r i z e d in that, for precipitating the blood sub¬ stance, mainly globin, the pH in said mixture is first adjusted to 7 to 8, or, in case that an agent promoting the separation is used, to 6.5 to 8, and thereafter the dehydrating agent, preferably ethanol, is added. 6. A method as claimed in claim 1, wherein a mix- ture of heme and blood substance obtained when cleaving hemoglobin is used as starting material, c h a r a c ¬ t e r i z e d in that, for precipitating the blood sub¬ stance, mainly globin, one or- more salts, such as sodium, potassium or ammonium chloride, sulphate, phosphate, citrate or tartrate are first added to said mixture, where¬ after the dehydrating agent is added and the pH is adjusted to over 6.0, or, in case that no agent promoting the separ¬ ation is added, to over 8.0. 7. A method as claimed in claims 1 to 6 , c h a r a c t e r i z e d in that the dehydrating agent is ethanol, methanol, ethylene glycol, glycerol, propanol, isopropanol, butanol or isobutanol, or a mixture thereof. 8. A method as claimed in claims 1 to 7, c h a r a c t e r i z e d .in that the compound in lb) is imidazole. 9. A method as claimed in claims 1 to 7, c h a r a c t e r i z e d in that the compound in lb) is a compound of the formula la, lb, or Ic: wherein the substituents have the following meaning: -R (R is a saturated or an unsaturated alkyl or aryl or CH ) , -H, -R (R) , -COOH, -R(COOH) , -COOR, -R(COOR) , -OH, -ROH, . -R(OH) n ,. -R(OH) n R,. -NH 2 , - (NH 2 ) n , -N 2 , - NR 2 , -N.-R, -CN, -R(CN) n , -Cl, -R(Cl) n , -F, - R (F) n . ~Br, -R(Br) n , -J, - R (J) n - -N0 2 , -R(N0 2 ) n , -COR, -R(COR) , -R(COCOOH) , -R(COCOOR) , -OCOR, -SH, -R(SH) n , -SR, -R(SR) ' , -S-S-, -S-CS-S-, -SO-H, -SO H, -RNHCOOH, -RNHCOOR, -R(NH) R(OH) , -R(NHCOR) COOH, -R(NHCOR) COOR, -NHCOOH, -NHCOOR, -R(NHCOOH) , -R(NHCOOR) . -R(NH„) (COOH) , 2 n n -R(NH ) (COOR) , -R(COOH) ' NHCOOH, -R(COOR) NHCOOR, z n n n n -RN(R)ROH, -R R(NH 2 ) n 0H7 n , -C0NH , -R(C0NH ) , -CONHNH„ , -R(CONHNH,,) , -NHCONH 2' 2 n 2 2 n -R(NHCONH 2 ) , -NHCOR, -RNHCOR, -CONHR, -RCONHR, -CO 3 , -R(CON 3 ) n , -RCOOHNHCORNH., , -S0 2 NHR, r S0 2 RNHCOOR, -CONHNHS0 2 R, -RS0 2 , -NHN0 2 , -RON0 2 , -RC1C00H,-RC1C00R, -RFCOOH, -RFCOOR, -RBrCOOH, -RBrCOOR, -RJCOOH, -RJCOOR, and wherein, in addition, X. and X 1 , and/or X- and X,- ' , ca be substituted by =0, =S, =CHR(N0 2 ) , =N(H 2 PO ) or =CHR, and for the imidazole compound lb, in addition, X. and X ' and/or X j . and X- 1 , and for the imidazoline compound Ic, moreover, X~ and X- ' , X, and X. ' , and/or X- and X ', also can be substituted by =0, =NH, =S, =CHR(N0 2 ) , =N(H 2 P0 4 ) a =CHR, in which substituent formulas n is a number from 1 to 5 , or any other similar compound, such as: Anserine NHCOCH 2 CH 2 NH 2 Carnosine SH _w . Ergotioneine -_ Histamine Imidazolyl-CH^CH NH. Histidole Imidazolyl-CH CH OH Dimethylhistamine Histidine Pilocarpine CH., Pilosinine SH C HN ' ^ NH, Thiolhistidine HC C-CH 2 CHCOOH Hercynine Imidazolyl-CH_C CHHCOO- N+ (CH 3 ) 3 Imidazolyl glycine Imidazolyl-CH (NH„) COOH Imidazolyl lactic acid Imidazolyl-CH 2 CHOHCOOH Imidazolyl propionic acid Imidazolyl-CH-CH-COOH Urocanic acid Imidazolyl-CH=CHCOOH Histamine-tyramine HOC 6 H 4 CH 2 CH 2 NHCH 2 CH 2 -i idazolyl Imidazolyl acidic acid Imidazolyl-CH-COOH Sulfaimidazoles Vitimidazole OH SH Thiolhistamine 10 A method as claimed in claims 1 to 7 , c h a r a c t e r i z e d in that the temperature is lowe than 0°C, preferably -5°C to -20°C. 11. A method as claimed in claims 1 to 8 , c h a r a c t e r i z e d in that the dehydrating agent is ethanol and in that it is present in the mixture in a concentration of over 40 %-by-volume, preferably 60 to 80 by-volume. 12. A he e-containing product, c h a r a c ¬ t e r i z e d in that it contains 15 to 55 % of heme and that it is prepared as defined in claims 1 to 11.

Inventors: LINDROOS P
Applicants: LINDROOS P
Application year: 1981
Main classification: A61K31
IPC codes: A23J1, A23L1, A61K31, A61K35, A61K38, A61P7, C07D487