329
Laboratory Animals (1979) 13,329-331
Haemolysis of various mammalian erythrocytes in sodium chloride, glucose and phosphate-buffer solutions TOSHIAKI
MATSUZAWA·
& YASUSHI IKARASHI
Japan Upjohn Research Laboratories, 168 Ohyagi, Takasaki, Gunma 370, Japan
Summary Mouse, rat, rabbit, hamster, cow, pig, sheep, guineapig, dog and human erythrocytes were studied. A 0·9% or stronger solution of sodium chloride completely prevented haemolysis; sheep and pig erythrocytes appeared the more fragile, while human and dog erythrocytes were not haemolized in concentrations of 0·4% or more. Haemolysis of human, rabbit, cow, hamster, guineapig, pig and sheep erythocytes was not observed in solutions of 0·4% or more of glucose. Except for sheep, human and dog erythrocytes, haemolysis was depressed in rate but not completely prevented by phosphate-butTer solution of pH 7·0. Most solutions used for the study of erythrocyte fragility contain sodium chloride, glucose or phosphates. The osmotic fragility of mammalian erythrocytes in hypotonic solutions of sodium chloride has been reported by Coldman, Gent & Good (1969) and Lewis & Ferguson (1966), but their fragility in glucose and phosphate buffer solutions has not been published. Materials and methods Human blood was collected from veins in the arms of a 33-year-old man. Blood samples from Hartely guineapigs, New Zealand White rabbits, IeR mice,
Sprague-Dawley
rats and golden hamsters were
obtained by cardiac puncture. Beagle dog blood was obtained by cephalic venepuncture. Holstein cow, Landrace pig and Dorset sheep blood was obtained from a slaughterhouse in Gunma, Japan. All blood samples were freshly collected in heparinized tubes. Blood was centrifuged to separate the plasma, which was removed by aspiration. Erythrocytes were washed 4 times with an equal volume of 0·9% sodium chloride solution. Sodium chloride and glucose solutions of varying concentrations and 1/15 M phosphate-buffer solutions of varying pH were used as test solutions. Osmotic pressure and pH of the solutions were measured with a vapour-pressure osmometer (model 5100A; Wescor
* Present
address: Yamanouchi Pharmaceutical Co. Ltd, Institute of Research and Development, Department of Toxicology and Safety Research, 1-8 Azusawa, Itabashi-ku, Tokyo 174, Japan.
Received
9 April 1979. Accepted 1 June 1979.
Inc., 459 South Main Street, Logan, Utah 84321, USA) and pH meter ('Expondomatic SS-2'; Beckman Instruments Inc., 2500 Harbor Boulevard, Fullerton, California 92634, USA), respectively. 10 ml of each solution tested was added to each of 3 centrifuge tubes. Tubes containing 10 ml of distilled water served as a reference colour for complete haemolysis. Exactly 0·1 ml of fresh difibrinated and aerated blood was added to all tubes, which were then incubated at 37°C for 30 min and centrifuged at 2500 rev/min for 10 min. The supernatant was decanted and the haemoglobin measured with a 'Hemoglobionometer' (Coulter Electronics Inc., 590 West 20th Street, Hialeah, Florida 33010, USA). The degree of haemolysis was calculated as a percentage of complete haemolysis. Results and discussion
Sodium chloride solution The corresponding fragility curves for the degree of haemolysis of erythrocytes of different species at different osmotic concentration is shown in Fig. I. The more fragile cells-sheep and pig-vary more with concentration than the less fragile dog and human erythrocytes: haemolysis of sheep and pig erythrocytes was prevented in a solution of 0·8% sodium chloride, with dog and human erythrocytes, 0·4%. The
osmotic fragility of cow, mouse, rabbit, hamster,
guineapig and rat erythrocytes was between these extremes. These data were in agreement with the results for each mammalian erythrocytes sample obtained by Coldman et at. (1969).
Glucose solution The degree of haemolysis of erythrocytes of different species in glucose solution of varying concentrations is shown in Fig. 2. Almost no haemolysis of human, rabbit, cow, hamster, guineapig and pig erythrocytes were observed in solutions of 0·4% or more in strength. Haemolysis of sheep erythrocytes was prevented in concentration of 0·6%. With dog, mouse and rat erythrocytes, as the concentration of glucose increased the degree of haemolysis decreased from 100% to about 2% haemolysis in 0·4% glucose solution, but then rose in much higher concentrations. Wurster & Shapiro (\963) reported the effect of sodium chloride solutions containing glucose on cow erythrocytes, which suggested that glucose did not
Downloaded from lan.sagepub.com at GEORGIAN COURT UNIV on March 6, 2015
330
Matsuzawa
& Ikarashi
100
Ul
'in
>-
"0
E ell
50
"0
E ell
50
-
'0
E 50
rabbit
'" .r::.
'"
"*
pig
cow rat __ mouse ..rabbit. hamster gUinea pig
human
o
.
5·4
6·0
7·0
8·0pH
Fig 3. The degree of haemolysis of erythrocytes of different species in phosphate buffer solutions
93% in solutions ranging from pH 5·4 to 8·0. Haemolysis of dog erythrocytes was also depressed in pH 7·0 solution, but increased rapidly in solutions of pH 7·2 to 8·0. Hammarlund & Pederson-Bjergaard (J 961) have reported haemolitic effects of acidic and
References Coldman, M. F. & Good, W. (1967). The distribution of sodium, potassium and glucose in the blood of some mammals. Comparative Biochemistry and Physiology 21, 201-206. Coldman, M. F., Gent, M. & Good, W. (1969). The osmotic fragility of mammalian erythrocytes in hypotonic solutions of sodium chloride. Comparative Biochemistry and Physiology 31,605-609.
Hamolyse von verschiedenen Saugetier-Erythrozyten PutTer-Losungen
alkali solutions on human erythrocytes which indicate that the effect of phosphated solutions is related to osmotic pressure. The osmotic pressure of 1/15 PBS in the present study was lower, and haemolysis was not prevented in any erythrocyte sample.
Hammarlund, E. R. & Pedersen-Bjergaard, K. (1961). Hemolysis of erythrocytes in various iso-osmotic solutions. Journal of Pharmaceutical Sciences 50,24-30. Lewis, J. H. & Ferguson, E. E. (1966). Osmotic fragility of premammalian erythrocytes. Comparative Biochemistry and Physiology 18,589-595. Wurster, D. E. & Shapiro, P. H. (1963). Investigation of the mechanism of urea-induced hemolysis. Journal of Pharmaceutical Sciences 52,33-38.
in Natriumchlorid-,
Glukose- und Phosphat-
T. MATSUZAWA & Y. IKARASHI Zusammenfassung Erythrozyten von Mausen, Ratten, Kaninchen, Hamstern, Kiihen, Schweinen, Schafen, Meerschweinchen, Hunden und Menschen wurden untersucht. Eine 0.9 %ige oder eine konzentriertere N atriumchlorid- Lasung verhinderte die Hiimolyse vollstiindig; Schaf- und Schweine-Erythrozyten schienen fUr Hamolyse am empfindlichsten zu sein, hingegen wurden Menschen- und Hunde-Erythrozyten bei Konzen-
trationen von 0.4% oder mehr nicht hiimolysiert. In 0.4 %igen oder konzentrierteren Glukose-Losungen wurde bei den Erythrozyten von Menschen, Kaninchen, Kiihen, Hamstern, Meerschweinchen, Schweinen und Schafen keine Hiimolyse beobachtet. Mit Ausnahme von Erythrozyten von Schafen, Menschen und Hunden konnte der Grad der Hamolyse durch eine Phosphat-PutTer-Lasung mit pH 7 zwar verringert, nicht aber vollstandig verhindert werden.
Downloaded from lan.sagepub.com at GEORGIAN COURT UNIV on March 6, 2015
Downloaded from lan.sagepub.com at GEORGIAN COURT UNIV on March 6, 2015
Laboratory Animals (1979) 13,329-331
Haemolysis of various mammalian erythrocytes in sodium chloride, glucose and phosphate-buffer solutions TOSHIAKI
MATSUZAWA·
& YASUSHI IKARASHI
Japan Upjohn Research Laboratories, 168 Ohyagi, Takasaki, Gunma 370, Japan
Summary Mouse, rat, rabbit, hamster, cow, pig, sheep, guineapig, dog and human erythrocytes were studied. A 0·9% or stronger solution of sodium chloride completely prevented haemolysis; sheep and pig erythrocytes appeared the more fragile, while human and dog erythrocytes were not haemolized in concentrations of 0·4% or more. Haemolysis of human, rabbit, cow, hamster, guineapig, pig and sheep erythocytes was not observed in solutions of 0·4% or more of glucose. Except for sheep, human and dog erythrocytes, haemolysis was depressed in rate but not completely prevented by phosphate-butTer solution of pH 7·0. Most solutions used for the study of erythrocyte fragility contain sodium chloride, glucose or phosphates. The osmotic fragility of mammalian erythrocytes in hypotonic solutions of sodium chloride has been reported by Coldman, Gent & Good (1969) and Lewis & Ferguson (1966), but their fragility in glucose and phosphate buffer solutions has not been published. Materials and methods Human blood was collected from veins in the arms of a 33-year-old man. Blood samples from Hartely guineapigs, New Zealand White rabbits, IeR mice,
Sprague-Dawley
rats and golden hamsters were
obtained by cardiac puncture. Beagle dog blood was obtained by cephalic venepuncture. Holstein cow, Landrace pig and Dorset sheep blood was obtained from a slaughterhouse in Gunma, Japan. All blood samples were freshly collected in heparinized tubes. Blood was centrifuged to separate the plasma, which was removed by aspiration. Erythrocytes were washed 4 times with an equal volume of 0·9% sodium chloride solution. Sodium chloride and glucose solutions of varying concentrations and 1/15 M phosphate-buffer solutions of varying pH were used as test solutions. Osmotic pressure and pH of the solutions were measured with a vapour-pressure osmometer (model 5100A; Wescor
* Present
address: Yamanouchi Pharmaceutical Co. Ltd, Institute of Research and Development, Department of Toxicology and Safety Research, 1-8 Azusawa, Itabashi-ku, Tokyo 174, Japan.
Received
9 April 1979. Accepted 1 June 1979.
Inc., 459 South Main Street, Logan, Utah 84321, USA) and pH meter ('Expondomatic SS-2'; Beckman Instruments Inc., 2500 Harbor Boulevard, Fullerton, California 92634, USA), respectively. 10 ml of each solution tested was added to each of 3 centrifuge tubes. Tubes containing 10 ml of distilled water served as a reference colour for complete haemolysis. Exactly 0·1 ml of fresh difibrinated and aerated blood was added to all tubes, which were then incubated at 37°C for 30 min and centrifuged at 2500 rev/min for 10 min. The supernatant was decanted and the haemoglobin measured with a 'Hemoglobionometer' (Coulter Electronics Inc., 590 West 20th Street, Hialeah, Florida 33010, USA). The degree of haemolysis was calculated as a percentage of complete haemolysis. Results and discussion
Sodium chloride solution The corresponding fragility curves for the degree of haemolysis of erythrocytes of different species at different osmotic concentration is shown in Fig. I. The more fragile cells-sheep and pig-vary more with concentration than the less fragile dog and human erythrocytes: haemolysis of sheep and pig erythrocytes was prevented in a solution of 0·8% sodium chloride, with dog and human erythrocytes, 0·4%. The
osmotic fragility of cow, mouse, rabbit, hamster,
guineapig and rat erythrocytes was between these extremes. These data were in agreement with the results for each mammalian erythrocytes sample obtained by Coldman et at. (1969).
Glucose solution The degree of haemolysis of erythrocytes of different species in glucose solution of varying concentrations is shown in Fig. 2. Almost no haemolysis of human, rabbit, cow, hamster, guineapig and pig erythrocytes were observed in solutions of 0·4% or more in strength. Haemolysis of sheep erythrocytes was prevented in concentration of 0·6%. With dog, mouse and rat erythrocytes, as the concentration of glucose increased the degree of haemolysis decreased from 100% to about 2% haemolysis in 0·4% glucose solution, but then rose in much higher concentrations. Wurster & Shapiro (\963) reported the effect of sodium chloride solutions containing glucose on cow erythrocytes, which suggested that glucose did not
Downloaded from lan.sagepub.com at GEORGIAN COURT UNIV on March 6, 2015
330
Matsuzawa
& Ikarashi
100
Ul
'in
>-
"0
E ell
50
"0
E ell
50
-
'0
E 50
rabbit
'" .r::.
'"
"*
pig
cow rat __ mouse ..rabbit. hamster gUinea pig
human
o
.
5·4
6·0
7·0
8·0pH
Fig 3. The degree of haemolysis of erythrocytes of different species in phosphate buffer solutions
93% in solutions ranging from pH 5·4 to 8·0. Haemolysis of dog erythrocytes was also depressed in pH 7·0 solution, but increased rapidly in solutions of pH 7·2 to 8·0. Hammarlund & Pederson-Bjergaard (J 961) have reported haemolitic effects of acidic and
References Coldman, M. F. & Good, W. (1967). The distribution of sodium, potassium and glucose in the blood of some mammals. Comparative Biochemistry and Physiology 21, 201-206. Coldman, M. F., Gent, M. & Good, W. (1969). The osmotic fragility of mammalian erythrocytes in hypotonic solutions of sodium chloride. Comparative Biochemistry and Physiology 31,605-609.
Hamolyse von verschiedenen Saugetier-Erythrozyten PutTer-Losungen
alkali solutions on human erythrocytes which indicate that the effect of phosphated solutions is related to osmotic pressure. The osmotic pressure of 1/15 PBS in the present study was lower, and haemolysis was not prevented in any erythrocyte sample.
Hammarlund, E. R. & Pedersen-Bjergaard, K. (1961). Hemolysis of erythrocytes in various iso-osmotic solutions. Journal of Pharmaceutical Sciences 50,24-30. Lewis, J. H. & Ferguson, E. E. (1966). Osmotic fragility of premammalian erythrocytes. Comparative Biochemistry and Physiology 18,589-595. Wurster, D. E. & Shapiro, P. H. (1963). Investigation of the mechanism of urea-induced hemolysis. Journal of Pharmaceutical Sciences 52,33-38.
in Natriumchlorid-,
Glukose- und Phosphat-
T. MATSUZAWA & Y. IKARASHI Zusammenfassung Erythrozyten von Mausen, Ratten, Kaninchen, Hamstern, Kiihen, Schweinen, Schafen, Meerschweinchen, Hunden und Menschen wurden untersucht. Eine 0.9 %ige oder eine konzentriertere N atriumchlorid- Lasung verhinderte die Hiimolyse vollstiindig; Schaf- und Schweine-Erythrozyten schienen fUr Hamolyse am empfindlichsten zu sein, hingegen wurden Menschen- und Hunde-Erythrozyten bei Konzen-
trationen von 0.4% oder mehr nicht hiimolysiert. In 0.4 %igen oder konzentrierteren Glukose-Losungen wurde bei den Erythrozyten von Menschen, Kaninchen, Kiihen, Hamstern, Meerschweinchen, Schweinen und Schafen keine Hiimolyse beobachtet. Mit Ausnahme von Erythrozyten von Schafen, Menschen und Hunden konnte der Grad der Hamolyse durch eine Phosphat-PutTer-Lasung mit pH 7 zwar verringert, nicht aber vollstandig verhindert werden.
Downloaded from lan.sagepub.com at GEORGIAN COURT UNIV on March 6, 2015
Downloaded from lan.sagepub.com at GEORGIAN COURT UNIV on March 6, 2015
