Haemolysis of various mammalian erythrocytes in sodium chloride

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
330
Matsuzawa
& Ikarashi
100
Ul
'in
>-
"0
E
ell
50
<0
.r::.
*'
o
o
0·1
0·2
03
0-4
0·5
06
0·7
08
0·9
,·0
% NaCI
8
39
72
107
145
178
209
236
272
290
315
mOs/kg
10
% glucose
Fig. 1. The degree of haemolysis of erythrocytes of different species in sodium chloride solutions
100
Ul
'in
>"0
E
ell
50
<0
.r::.
*'
o
o
2
4
6
8
Fig. 2. The degree of haemolysis of erythrocytes of different species in glucose solutions
affect the cell membrane directly. Glucose concentrations in the serum and blood cells of various
mammals was reported by Coldman & Good (1967),
but there is no relationship between glucose concentration in blood cells and the degree of haemolysis in
the present study. The fragility of dog, rat and mouse
erythrocytes in glucose solutions may be related to the
nature of the cell membrane.
1/15 phosphate buffer solution (PBS)
Fig. 3 shows the relationship between changes in pH
and percent haemolysis for each mammalian erythrocyte sample. Except for sheep erythrocytes,haemolysis was depressed in rate but not completely
suppressed in solution with a pH of 7·0. No human
erythrocytes were haemolyzed in solutions of pH 6,0
or higher. Haemolysis of sheep erythrocytes was 98 to
Mammalian
100
erythrocyte
haemolysis in vitro
rat
sheep
331
guinea pig
sheep
pig
cow
dog
til
'iii
>-
'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.