Biomaterials, Artificial Cells and Immobilization Biotechnology
ISSN: 1055-7172 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ianb18
Stabilized Hemoglobins as Acellular Resuscitative Fluids L. C. Cerny, A. Green, B. Noga & E. R. Cerny To cite this article: L. C. Cerny, A. Green, B. Noga & E. R. Cerny (1992) Stabilized Hemoglobins as Acellular Resuscitative Fluids, Biomaterials, Artificial Cells and Immobilization Biotechnology, 20:2-4, 327-330, DOI: 10.3109/10731199209119651 To link to this article: http://dx.doi.org/10.3109/10731199209119651
Published online: 11 Jul 2009.
Submit your article to this journal
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Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ianb18 Download by: [McMaster University]
Date: 17 March 2016, At: 00:15
BIOHAT., ART. CELLS & IHHOB. BIOTECH., 2 0 ( 2 - 4 ) , 327-330 (1992)
STABILIZED HEMOGLOBINS AS ACELLULAR RESUSCITATIVE FLUIDS
Downloaded by [McMaster University] at 00:15 17 March 2016
L.C. Cerny, A. Green, B. Noga Utica College of Syracuse University and E.R. Cerny Cernyland of Utica 13502 Utica, NY ABSTRACT This study reports some recent work dealing with the stabilization of the tetramers of hemoglobin. It is shown that by using a variety of diacids, it is possible to increase the P50 above that of stroma free hemoglobin. In order to lengthen the retention times in the circulatory system, the stabilized hemoglobins were complexed with both hydroxyethyl starch polymers and polyol tetronic polymers. The resulting hemoglobin-polymer compounds were then freeze-dried. It was possible to reconstitute the powder by the addition of physiological saline when needed. The methods presented here appear to be appear to be as effective as using pyridoxal phosphate but at a fraction of the cost. MATERIALS AND METHODS Hemoqlobin. The hemoglobin (Hgb) was prepared from out-dated blood. In an effort to scale-up this process, reduce the costs and increase the Hgb concentration, preliminary results indicate that the Boycott Effect can be employed to accomplish this goal (1). This technique is detailed by Davis in his thesis on super settlers (2). On a laboratory scale, it essentially takes $ large graduated cylinder, 1000 to 2000 ml, which is inclined at 4 5 to the horizon by a clamp attached to a ringstand. The whole blood samples are carefully poured into the cylinder and allowed to settle in the cold, 4 C. Usually the process is allowed to take place overnight, however after about 5 hours, the red cells are firmly packed. The plasma layer and buffy coat are removed. The red cells are then hemolyzed with 1.25 volume of distilled water and 0 . 5 volume of toluene is added. The resulting mixture is either shaken or stirred until the process is complete. It is then transferred to another graduated cylinder at a 45O inclination and maintained at 4 C overnight. The resulting organic layer is removed and the Hgb solution is dialyzed in tubing against distilled H20 ( 1 : 4 ) . It has been shown that because of the reduced amount of handling and transferring of the solutions, the concentration of the Hgb is almost doubled and the methemoglobin concentration is halved. The extraction of the Hgb from the red cells by hemolysis with water and toluene produces a Hgb solution basically free of stroma. To expedite the purification of the hgb solution, it is initially filtered through a series of Whatman papers from coarse to fine degrees of porosity, then finally through 0.221(m filter to render it sterile.
321
COPyriJlt
6 1992 by Mucel Dekkar. Inc.
CERNY ET AL.
328
UBLLL
Downloaded by [McMaster University] at 00:15 17 March 2016
STABILIZED HEMOGLOBIN Oraanic Acid
Structure
Oxalic
HOOC-COOH
Malonic
nooc-cH2-coon
Succinic
IlOOC -CII
Glutaric
IIOOC-C~~-CH~-CH~-COO~
Adipic
HOOC-CH~-CH~-CH~-CH~-COO~
Pripol 1009
D i m e r Fatty Acid
2-CH 2-COOH
?n2 -
no - cI -
Citric
M.W.
560
COOH
COOH CH2 - COOH
% Saturation = KP”
1 1
+
RPn
n -
-R
P50(-9
H9b
2.21
0.00218
16
Hgb-oxalic
2.52
0.0000527
49.5
Hgb-Malonic
1.54
0.00234
51 . O
Hgb-succinic
1.98
0.000326
51.2
Hgb-glutaric
2.02
0.000416
44
Hgb-adipic
2.24
0.000064 1
75
Hgb-Fatty acid
2.29
0.000155
45.9
H g b-c i tr ic
1.22
0.00831
51.2
Hgb-XL (Benesch)
1.58
0.0024
46.1
1
TABLE I1
Tetronic
707 1307 908
Molecular Weight
Typical P r o m r t i e s Melting Acute Oral Acute Dermal Point C LD50 G/Kg LD50 G/Kg
- 12,000 18,600 27,000
49 54 58
15 15 15
3 3 3
Irritation Skin Eye Mild Min. Mild
Mild
Hin. Mild
Downloaded by [McMaster University] at 00:15 17 March 2016
STABILIZED HEMOGLOBINS AS ACELLULAR RESUSCITATIVE FLUIDS
319
Tetronic PolYols. The recent success of producing a trialdehyde polymer from hydroxyethyl starch (HES) was encouraging ( 3 ) . It was anticipated that if four active sites could be attained, then the possibility of complexing the hemoglobin would be further enhanced. The approach to the problem was slightly altered. A series of block polymers were developed in the early 1950's by the BASF Wyandotte Corporation (4). The polymers have the commercial trade name, Tetronic Polyols. The materials are formed by the sequential addition of both ethylene and propylene oxides to a fourpronged molecule such a s ethylene diamine. Three of these Tetronic Polyols were tried. The physical chemical properties are shown in Table 11. The fact that these polymers have a homogeneous molecular weight and a definite melting point make them attractive starting materials. The Tetronic Polyols apparently undergo all the typical reactions of alcohols. The toxicity levels of these three compounds are also presented in Table 11. According to generally accepted standards, the Tetronic polyols are considered non-toxic and nonhazardous. Tetronic Modification. The Tetronic Polyol was used in the synthesis in two forms. The first method incorporated the polymer in its unmodified state as a component with available alcohol groups. In the second technique, the four alcohols were converted to the aldehyde moiety by the mild oxidation of Pfitzner and Moffatt (5). A typical reaction dissolved twenty grams of the Tetronic Polyol in 125 ml of dimethyl sulfoxide. Then about 0.75 gm of dicyclohexylcarbodiimide was added along with 0.1 gm of phosphoric acid. The reaction mixture was stirred for 12 hours at room temperature. If crystals of DCC-urea were formed, they were removed by filtration. The remaining liquid was dialyzed against distilled water at 4 C. Infrared spectra was used to follow the conversion of an alcohol to an aldehyde. Hemoqlobin Modifications. The tetramers of Hgb were stabilized and modified using glutaric acid, oxalic acid and citric acid. As indicated in Table I, these diacids are effective in stabilizing the Hgb and maintaining a satisfactory p o. The basic method for this reaction has been previously reportea ( 3 ) . Hemoqlobin-Tetronic Complexes. An oxygen-transporting reSUSCit.3tive fluid was synthesized by starting with either the alcohol form of the Tetronic Polyol or its aldehyde moiety. These synthetic routes will follow those which were previously used to produce Hgb complexes with modified hydroxyethyl starches (HES) (6). CONCLUSIONS In summary it can be tentatively concluded that it is possible to form a variable resuscitative fluid from a Tetronic Polyol and a modified hemoglobin. Since these polyols are considered both non-toxic and non-hazardous and the fact that one of the compounds which has been used to modify the hemoglobin, glutaric acid, has been used as a food adiculant, provides encouragement. The polyols are readily available commercially and can be synthesized with a wide range of molecular weights. This added dimension will permit the formation of complexes, not only with the molecular weight as a parameter, but also a control on the circulation retention time by using blends. REFERENCES 1) Falvo, F . et al, Biorheology B:465-71
(1979)
2) Davis, R.H., "The Operation and Stability of Continuous Inclined Supersettlers," Ph.D. Thesis, Stanford University, Stanford, CA 1983 3 ) Cerny, L.C.
et al, Clin. Hemorheology 8:621-36 (1988)
330
CERNY ET AL.
4) Technical data on Tetronic Polyols Series of nonionic surfactants.
BASF Wyandotte Corp., Wyandotte, MI 48192 5) Pfitzner, K.E. (1965)
Downloaded by [McMaster University] at 00:15 17 March 2016
6) Cerny, L.C.
and Moffatt, J . G .
J. Am. Chem.
et al, Clin. Hemorheol. 2:355-65
SOC.
(1982)
=:5661-70
ISSN: 1055-7172 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ianb18
Stabilized Hemoglobins as Acellular Resuscitative Fluids L. C. Cerny, A. Green, B. Noga & E. R. Cerny To cite this article: L. C. Cerny, A. Green, B. Noga & E. R. Cerny (1992) Stabilized Hemoglobins as Acellular Resuscitative Fluids, Biomaterials, Artificial Cells and Immobilization Biotechnology, 20:2-4, 327-330, DOI: 10.3109/10731199209119651 To link to this article: http://dx.doi.org/10.3109/10731199209119651
Published online: 11 Jul 2009.
Submit your article to this journal
Article views: 1
View related articles
Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ianb18 Download by: [McMaster University]
Date: 17 March 2016, At: 00:15
BIOHAT., ART. CELLS & IHHOB. BIOTECH., 2 0 ( 2 - 4 ) , 327-330 (1992)
STABILIZED HEMOGLOBINS AS ACELLULAR RESUSCITATIVE FLUIDS
Downloaded by [McMaster University] at 00:15 17 March 2016
L.C. Cerny, A. Green, B. Noga Utica College of Syracuse University and E.R. Cerny Cernyland of Utica 13502 Utica, NY ABSTRACT This study reports some recent work dealing with the stabilization of the tetramers of hemoglobin. It is shown that by using a variety of diacids, it is possible to increase the P50 above that of stroma free hemoglobin. In order to lengthen the retention times in the circulatory system, the stabilized hemoglobins were complexed with both hydroxyethyl starch polymers and polyol tetronic polymers. The resulting hemoglobin-polymer compounds were then freeze-dried. It was possible to reconstitute the powder by the addition of physiological saline when needed. The methods presented here appear to be appear to be as effective as using pyridoxal phosphate but at a fraction of the cost. MATERIALS AND METHODS Hemoqlobin. The hemoglobin (Hgb) was prepared from out-dated blood. In an effort to scale-up this process, reduce the costs and increase the Hgb concentration, preliminary results indicate that the Boycott Effect can be employed to accomplish this goal (1). This technique is detailed by Davis in his thesis on super settlers (2). On a laboratory scale, it essentially takes $ large graduated cylinder, 1000 to 2000 ml, which is inclined at 4 5 to the horizon by a clamp attached to a ringstand. The whole blood samples are carefully poured into the cylinder and allowed to settle in the cold, 4 C. Usually the process is allowed to take place overnight, however after about 5 hours, the red cells are firmly packed. The plasma layer and buffy coat are removed. The red cells are then hemolyzed with 1.25 volume of distilled water and 0 . 5 volume of toluene is added. The resulting mixture is either shaken or stirred until the process is complete. It is then transferred to another graduated cylinder at a 45O inclination and maintained at 4 C overnight. The resulting organic layer is removed and the Hgb solution is dialyzed in tubing against distilled H20 ( 1 : 4 ) . It has been shown that because of the reduced amount of handling and transferring of the solutions, the concentration of the Hgb is almost doubled and the methemoglobin concentration is halved. The extraction of the Hgb from the red cells by hemolysis with water and toluene produces a Hgb solution basically free of stroma. To expedite the purification of the hgb solution, it is initially filtered through a series of Whatman papers from coarse to fine degrees of porosity, then finally through 0.221(m filter to render it sterile.
321
COPyriJlt
6 1992 by Mucel Dekkar. Inc.
CERNY ET AL.
328
UBLLL
Downloaded by [McMaster University] at 00:15 17 March 2016
STABILIZED HEMOGLOBIN Oraanic Acid
Structure
Oxalic
HOOC-COOH
Malonic
nooc-cH2-coon
Succinic
IlOOC -CII
Glutaric
IIOOC-C~~-CH~-CH~-COO~
Adipic
HOOC-CH~-CH~-CH~-CH~-COO~
Pripol 1009
D i m e r Fatty Acid
2-CH 2-COOH
?n2 -
no - cI -
Citric
M.W.
560
COOH
COOH CH2 - COOH
% Saturation = KP”
1 1
+
RPn
n -
-R
P50(-9
H9b
2.21
0.00218
16
Hgb-oxalic
2.52
0.0000527
49.5
Hgb-Malonic
1.54
0.00234
51 . O
Hgb-succinic
1.98
0.000326
51.2
Hgb-glutaric
2.02
0.000416
44
Hgb-adipic
2.24
0.000064 1
75
Hgb-Fatty acid
2.29
0.000155
45.9
H g b-c i tr ic
1.22
0.00831
51.2
Hgb-XL (Benesch)
1.58
0.0024
46.1
1
TABLE I1
Tetronic
707 1307 908
Molecular Weight
Typical P r o m r t i e s Melting Acute Oral Acute Dermal Point C LD50 G/Kg LD50 G/Kg
- 12,000 18,600 27,000
49 54 58
15 15 15
3 3 3
Irritation Skin Eye Mild Min. Mild
Mild
Hin. Mild
Downloaded by [McMaster University] at 00:15 17 March 2016
STABILIZED HEMOGLOBINS AS ACELLULAR RESUSCITATIVE FLUIDS
319
Tetronic PolYols. The recent success of producing a trialdehyde polymer from hydroxyethyl starch (HES) was encouraging ( 3 ) . It was anticipated that if four active sites could be attained, then the possibility of complexing the hemoglobin would be further enhanced. The approach to the problem was slightly altered. A series of block polymers were developed in the early 1950's by the BASF Wyandotte Corporation (4). The polymers have the commercial trade name, Tetronic Polyols. The materials are formed by the sequential addition of both ethylene and propylene oxides to a fourpronged molecule such a s ethylene diamine. Three of these Tetronic Polyols were tried. The physical chemical properties are shown in Table 11. The fact that these polymers have a homogeneous molecular weight and a definite melting point make them attractive starting materials. The Tetronic Polyols apparently undergo all the typical reactions of alcohols. The toxicity levels of these three compounds are also presented in Table 11. According to generally accepted standards, the Tetronic polyols are considered non-toxic and nonhazardous. Tetronic Modification. The Tetronic Polyol was used in the synthesis in two forms. The first method incorporated the polymer in its unmodified state as a component with available alcohol groups. In the second technique, the four alcohols were converted to the aldehyde moiety by the mild oxidation of Pfitzner and Moffatt (5). A typical reaction dissolved twenty grams of the Tetronic Polyol in 125 ml of dimethyl sulfoxide. Then about 0.75 gm of dicyclohexylcarbodiimide was added along with 0.1 gm of phosphoric acid. The reaction mixture was stirred for 12 hours at room temperature. If crystals of DCC-urea were formed, they were removed by filtration. The remaining liquid was dialyzed against distilled water at 4 C. Infrared spectra was used to follow the conversion of an alcohol to an aldehyde. Hemoqlobin Modifications. The tetramers of Hgb were stabilized and modified using glutaric acid, oxalic acid and citric acid. As indicated in Table I, these diacids are effective in stabilizing the Hgb and maintaining a satisfactory p o. The basic method for this reaction has been previously reportea ( 3 ) . Hemoqlobin-Tetronic Complexes. An oxygen-transporting reSUSCit.3tive fluid was synthesized by starting with either the alcohol form of the Tetronic Polyol or its aldehyde moiety. These synthetic routes will follow those which were previously used to produce Hgb complexes with modified hydroxyethyl starches (HES) (6). CONCLUSIONS In summary it can be tentatively concluded that it is possible to form a variable resuscitative fluid from a Tetronic Polyol and a modified hemoglobin. Since these polyols are considered both non-toxic and non-hazardous and the fact that one of the compounds which has been used to modify the hemoglobin, glutaric acid, has been used as a food adiculant, provides encouragement. The polyols are readily available commercially and can be synthesized with a wide range of molecular weights. This added dimension will permit the formation of complexes, not only with the molecular weight as a parameter, but also a control on the circulation retention time by using blends. REFERENCES 1) Falvo, F . et al, Biorheology B:465-71
(1979)
2) Davis, R.H., "The Operation and Stability of Continuous Inclined Supersettlers," Ph.D. Thesis, Stanford University, Stanford, CA 1983 3 ) Cerny, L.C.
et al, Clin. Hemorheology 8:621-36 (1988)
330
CERNY ET AL.
4) Technical data on Tetronic Polyols Series of nonionic surfactants.
BASF Wyandotte Corp., Wyandotte, MI 48192 5) Pfitzner, K.E. (1965)
Downloaded by [McMaster University] at 00:15 17 March 2016
6) Cerny, L.C.
and Moffatt, J . G .
J. Am. Chem.
et al, Clin. Hemorheol. 2:355-65
SOC.
(1982)
=:5661-70
