Hemoglobin Function in Stored Blood:
VI. The Effect of Phosphate on Erythrocyte ATP and 2,3-DPG
M A J . R. BEN DAWSON, JR., MC,
AND WALTER F. KOCHOLATY, P H . D .
Mood Transfusion Division and Biochemistry Division, US Army Medical Research Labortory,
Fort Knox, Kentucky 40121
ABSTRACT
Dawson, R. Ben, and Kocholaty, Walter F.: Hemoglobin function in stored
blood. VI. The effect of phosphate on erythrocyte ATP and 2,3-DPG. Amer.
J. Clin. Path. 56: 656-660, 19711. Inorganic phosphate, known to stimulate
erythrocyte glycolysis, is present in one of the preservatives for blood storage,
citrate-phosphate-dextrose (CPD), but not the other, acid-citrate-dextrose
(ACD). Both ACD and CPD were developed before it was known that erythrocyte 2,3-diphosphoglycerate (2,3-DPG) was necessary for normal hemoglobin
function. The phosphate in CPD gives a final concentration of 2 mM, which
is less than the concentrations originally used to study the phosphate effect.
Therefore, the present study was designed to see whether higher concentrations of phosphate would better maintain erythrocyte concentrations of 2,3DPG during blood storage. For maintaining 2,3-DPG, 2 mM phosphate was
the best; however, 5 mM phosphate was nearly as good. Ten, 15, and 20 mM
phosphate were progressively worse. Also, there was very little difference
between 2 and 5 mM phosphate with respect to ATP, and the highest phosphate concentration was the worst.
henceforth referred
to as "phosphate," is known to stimulate
the metabolism of glucose in human erythrocytes. The mechanism of the phosphate
effect has been reviewed in recent studies,10- 12 and will not be discussed further in
this report. Recent experiments from this
laboratory 4 • have indicated that slightly
higher concentrations of adenosine triphosphate (ATP) are maintained during storage when the preservative contains phosphate, 2 mM, as in citrate-phosphate-dextrose (CPD). In the present experiments
higher concentrations of phosphate have
been used in an attempt to determine
INORGANIC PHOSPHATE,
Received October 8, 1970; revised manuscript received January 15, 1971; accepted for publication
January 29, 1971.
• Reference 4 is the fifth paper in this series.
whether the amount of phosphate in CPD
is optimal for maintenance of 2,3-diphosphoglycerate (2,3-DPG) as well as ATP
during storage.
Methods
Blood was drawn from a single healthy
young male volunteer into identical plastic
bags (Fenwal) containing the preservatives
to be compared (Table 1). T h e preservatives were sterilized by millipore filtration
(0.22 p pore width). The five bags were collected and stored together at 4 ± 1 C. in a
blood bank refrigerator. At weekly intervals aliquots were removed for study. Anaerobic and aseptic technics were used during
collection, storage, and sampling.
Concentrations of 2,3-DPG and ATP
were determined by the enzymic recycling
656
November
1971
Hb FUNCTION IN STORED BLOOD: PO, EFFECT
657
Table 1. CPD Anticoagulants with Various Phosphate Concentrations
(pH 5.50 ± 0.004)»
Trisodium citrate—2 H20
Citric acid—H20
Dextrose—H2O
NaHjPOi—HaO
Total citrate
Phosphate
Phosphate
2mMt
5mMf
Phosphate
lOmMf
2.800 Gm.
0.392 Gm.
2.450 Gm.
0.222 Gm.
2.153 Gm.
2.856 Gm.
0.352 Gm.
2.450 Gm.
0.555 Gm.
2.153 Gm.
2.912 Gm.
0.333 Gm.
2.450 Gm.
1.110 Gm.
2.172 Gm.
4
Phosphate
Phosphate
15mMt
20mMf
2.968 Gm.
0.325 Gm.
2.450 Gm.
1.665 Gm.
2.201 Gm.
3.192 Gm.
0.288 Gm.
2.450 Gm.
2.220 Gm.
2.312 Gm.
* Amounts given are per 100 ml. of anticoagulant.
t Final concentration of inorganic phosphate which will be present after the blood is collected into the anticoagulant.
method of Lowry,9 using ratio fluorometry
as described by Bunn and associates.1 The
mean concentration of 2,3-DPG for fresh
blood specimens from a normal population
(n = 18) was 4.55 ±0.12 (S.E.) ,M per ml.
of erythrocytes, and the mean A T P concentration was 1.34 ±0.033 (S.E.) ^M per
ml. of erythrocytes.
Oxygen dissociation curves were performed on whole blood by spectrophotometry (Instrumentation Laboratories Model
No. 182 Co-Oximeter) after equilibration
in a tonometer (IL No. 137) at 37 C. The
gas mixtures had a partial pressure of CO a
of 40 mm. Hg and various proportions of
oxygen and nitrogen. The pH of each
equilibrated sample was measured anaerobically at 37 C. The partial pressure of oxygen, p 0 2 , of the mixtures was measured by
amperometric blood gas analysis, and was
corrected to the whole blood value at 7.40
by the Severinghaus nomogram. 11 The IL
No. 113 pH/Blood Gas Meter was used.
Oxygen dissociation curves were drawn
from points determined by three to five
equilibrations and these data were graphed
logarithmically according to the Hill equation, as described by Bunn. 1 T h e value of
p 0 2 at which hemoglobin is 50% saturated
with oxygen was determined and designated P 60 . This is a direct, although inverse, measure of oxygen affinity. The P6o
value for 11 fresh blood samples collected
into ACD from healthy volunteers was 22.7
± 0.41 (S.E.) mm. Hg. This is midway between the values reported by Gullbring
and Strom 8 and by Bunn and colleagues,1
25.7 ± 0.35 (S.E.) for fresh ACD blood.
Plasma hemoglobin was determined by
the method of Crosby and Furth. 3
Results
In Figure 1, A T P concentrations during
storage of blood preserved in CPD with
various amounts of phosphate are shown.
A difference becomes apparent after the
second week, when the A T P concentration
at 20 mM of phosphate falls progressively
below the levels seen with the other concentrations. T h e 2 to 15 mM concentrations seemed to provide for similar concentrations of A T P after the second week, all
values being above those seen for the 20
mM phosphate solution.
Concentrations of 2,3-diphosphoglycerate
during storage of blood preserved in CPD
with various amounts of phosphate are
seen in Figure 2. T h e lowest concentration,
2 mM (CPD), provides for the best maintenance of 2,3-DPG during the first two
weeks of storage. T h e next best concentration is 5 mM, with 10 mM being intermediate. T h e 15 and 20 mM phosphate
concentrations are seen to be associated
with a rapid decrease in 2,3-DPG concentrations in the first week of storage.
658
A.J.C.P.—Vol. 56
DAWSON AND KOCHOLATY
* 4 \ \ NX
2
3
WEEKS OF STORAGE
WEEKS OF STORAGE
19-
o
J3
X
M
-O
T
o
1817-
B!'
16-
/•
3
1
1
1
2
1
3
1
4
1
5
^M 2,3-DPG/ml RBC
1
7
3
WEEKS OF STORAGE
KEY FOR FIGURES 1, 2, and 4. Concentrations of. PCv 2 mM = open triangles; 5 mM = solid triangles; 10 mM = open diamonds; 15 mM = solid squares; 20 mM = solid circles.
FIG. 1 (upper left). Concentrations of A T P during storage of blood preserved in CPD of various PO, concentrations.
FIG. 2 (upper right). Concentrations of 2,3-DPG during storage of blood preserved in CPD
of various PO, concentrations.
FIG. 3 (lower left). Relationship between 2,3-DPG and P r a after seven days of storage in CPD
with various amounts of phosphate.
FIG. 4 (lower right). Amount of spontaneous hemolysis during storage of blood preserved in
CPD of various P 0 4 concentrations.
In Figure 3 a correlation between 2,3DPG and P 50 , an expression of hemoglobin
function, is seen.
Figure 4 shows the amount of spontaneous hemolysis occurring during storage of
blood preserved with various concentrations of phosphate. Except for one aberrant point, CPD (2 mM) at 3 weeks, it
seems clear that the greatest amount of
hemolysis after 2 weeks occurs with the
two highest phosphate concentrations, 15
mM and 20 mM. The lower concentrations, 5 mM and 10 mM, show minimal
spontaneous hemolysis through 3 to 4
weeks of storage.
The pH values (Table 2) in CPD stored
with various amounts of phosphate show
a gradual decrease in pH during a 5-week
storage period, as expected. In the low
phosphate preservatives, 2 mM and 5 mM,
November 1971
659
Hb FUNCTION IN STORED BLOOD: P0 4 EFFECT
Table 2. Blood pH Values in CPD with Various Amounts of Phosphate
CPD
ODays
7 Days
14 Days
21 Days
28 Days
35 Days
2mM
S mM
10 mM
15 mM
20 mM
7.312
7.309
7.228
7.105
7.073
7.140
7.128
7.033
7.003
7.005
7.040
7.039
7.031
6.923
6.930
6.991
6.941
6.912
6.860
6.849
6.942
6.900
6.914
6.846
6.837
6.903
6.867
6.873
6.829
6.815
the pH at day 0 is approximately 0.2 pH
units higher than that of the high phosphate preservatives, 15 mM and 20 mM.
This difference decreases to approximately
0.15 pH units at 7 days and 0.1 pH units
at 14 days. From 21 to 35 days the difference between the low phosphate preservatives and high phosphate preservatives is
usually less than 0.1 pH units.
Discussion
The studies reported here represent a
further attempt to establish the optimal
conditions for storage of whole blood in a
liquid state that will maintain 2,3-DPG as
well as ATP. Both acid-citrate-dextrose
(ACD) and citrate-phosphate-dextrose
(CPD) were developed before it was known
that 2,3-DPG was necessary for normal hemoglobin function. Previous publications
from this laboratory have established that
2,3-DPG is maintained better when the
blood is stored in CPD than when stored
in ACD. 5-7 In a recent report, we presented evidence that the higher pH of
CPD is the reason for its maintenance of
2,3-DPG levels during storage.4 It was also
shown in that report that whenever phosphate was present in the preservative,
whether at the pH of ACD or that of CPD,
ATP concentrations were better maintained. Previous studies using inorganic
phosphate as a supplement for the storage
of human erythrocytes have used 10 mM
phosphate. 2
From the present study, it seems clear
that the low amount of phosphate is optimal for maintaining 2,3-DPG and A T P
during storage of blood in a liquid state at
4 C. For maintaining 2,3-DPG, 2 mM phosphate was best; however, 5 mM phosphate
was nearly as good (Fig. 2). Also, there
seemed to be very little difference between
2 and 5 mM phosphate with respect to
A T P concentrations, and spontaneous hemolysis was less with the lower phosphate
concentrations (Fig. 4). The greatest spontaneous hemolysis occurred with 15 and
20 mM phosphate. In Figure 3, the wellknown correlation between 2,3-DPG concentrations of the erythrocyte and P 50 , a
measure of hemoglobin function, is shown.
This, of course, indicates that whenever
2,3-DPG concentrations are maintained at
normal levels hemoglobin function will be
normal. Also, with decreasing 2,3-DPG concentrations, as during storage, a decrease
in P 50 from normal values (see Methods),
indicating impairment of hemoglobin function, will occur.
It would appear from the pH values
given in Table 2 that the low phosphate
preservatives, 2 and 5 mM, provided a
more effective buffering capacity than the
high phosphate preservatives because the
initial or day 0 pH values are more than
0.2 pH units higher. This is an important
difference when one realizes that the preservatives, listed in Table 1, have the same
pH before blood is collected into them. It
660
DAWSON AND KOCHOLATY
is also important to realize that the difference in pH between blood collected into
ACD and CPD, which was given to explain
the better maintenance of 2,3-DPG in CPD,
is usually 0.2 pH units.4 Although we may,
in part, be studying a pH effect in the present experiments, it would seem that higher
levels of inorganic phosphate than are
present in prepared CPD, that is, 2 mM
phosphate, would offer no advantage.
Acknowledgment. John L. Gray, Thomas J. Ellis,
Edith B. Ledford, and Thomas A. Billings gave
technical assistance.
References
1. Bunn HF, May MH, Kocholaty WF, et al:
Hemoglobin function in stored blood. J Clin
Invest 48:311-321. 1969
2. Chanutin A: Effect of storage of blood in ACDadenine-inorganic phosphate plus nucleosides
on metabolic intermediates of human red
cells. Transfusion 7:409-419, 1967
3. Crosby WH, Furth FW: A modification of the
benzidine method for measurement of hemoglobin in plasma and urine. Blood 11:380383, 1956
4. Dawson RB, Kocholaty WF, Gray JL: The
hemoglobin function and 2,3-DPG levels of
blood stored at 4 C in ACD and CPD: The
pH effect. Transfusion 10:299-304, 1970
r
A.J.C.P.—Vol. 56
i. Dawson RB Jr: The hemoglobin function of
blood stored at 4 C in ACD and CPD (Abstract). Clin Res 17:323, 1969
6. Dawson RB Jr, Kocholaty WF, Shields CE, et
al.: The control of hemoglobin function during storage of blood at 4 C, Red Cell Metabolism and Function. Edited by GJ Brewer.
New York, Plenum Press, 1970, pp 305-317
USAMRL Report No. 836, Fort Knox, Kentucky, 1 October 1969)
7. Dawson RB Jr, Kocholaty WF, Ellis TJ, et al.:
The control of hemoglobin function in blood
stored for transfusion purposes, Blood Oxygenation. Edited by D Hershey. New York,
Plenum Press, 1970, pp 231-242 (USAMRL
Report No. 856, Fort Knox, Kentucky, 1970)
8. Gullbring B, Strom G: Changes in oxygencarrying function of human hemoglobin during storage in cold acid-citrate-dextrose solution. Acta Med Scand 155:413-430, 1956
9. Lowry O. H., Passonneau JV, Hasselberger FX,
et al.: Effect of ischemia on known substrates
and cofactors of the glycolytic pathway in
brain. J Biol Chem 239:18-30, 1964
10. Rose LA, Warms JVB, O'Connell EL: Role of
inorganic phosphate in stimulating the glucose utilization of human red blood cells. Biochem Biophys Res Commun 15:33-37, 1964
11. Severinghaus TW: Oxyhemoglobin dissociation
curve correction for temperature and pH in
human blood. J Appl Physiol 12:485-486,
1958
12. Tsuboi KK, Fukunaga K: Inorganic phosphate
and enhanced glucose degradation by the intart erythrocyte. J Biol Chem 240:2806-2810,
1965