PHOSPHATASES I N BLOOD OF MAN
VALUES IN WHOLE BLOOD, PLASMA, CYTOLYSATES AND
ERYTHROCYTIC SUSPENSIONS*
H.BEHRENDT,M.D.
From the Department of Pediatrics, New York Post-Graduate
and Hospital, New York, New York
Medical
School
The results of previously reported observations2 indicate that human erythrocytes contain primarily "acid" phosphomonoesterase with an optimal activity at
pH 4.9-5.0, while "alkaline" phosphatase, with an optimum at 9.0, is present
only in very small amounts. The reverse is true of normal human plasma, in
which alkaline phosphatase activity is high as compared with acid phosphatase
activity. The predominance of acid phosphatase activity in red cells and of
alkaline phosphatase activity in plasma can be demonstrated with either glycerophosphate or phenylphosphate serving as substrate. The measurements made
by King, Wood, and Delory15 confirmed these findings in principle. The absence
of a magnesium effect and the weakness of fluoride inhibition, as established for
the red cell phosphatase at pH 4.9, corroborated the suggestion made by Sullivan,
Gutman, and Gutman 21 that the acid enzymes of red cells and plasma are not
identical.
All estimations of phosphatase activity of erythrocytes reported so far were
carried out on washed and subsequently hemolyzed red cells. The studies to
be reported here were made on whole blood, blood plasma, blood cytolysates
(hemolyzed erythrocytes), and suspensions of erythrocytes in saline solution.
With few exceptions, measurements were made only at the established optimum
pH of the acid and alkaline enzymes.
PLASMA AND CYTOLYSATES
Venous blood was withdrawn into tubes containing enough dried sodium oxalate solution
or heparin to prevent clotting. A portion of this blood sample was transferred into a
hematocrit tube for estimation of the relative cell volume. I n t h e remaining portion,
plasma and cells were separated by centrifligation; as-much plasma as possible was pipetted
off and the remaining red cells were washed three times with phj'siologic saline solution.
T h e washed cells obtained from A ml. of whole blood were hemolyzed by adding distilled
water to bring t h e total volume of t h e eytolysate to A ml., or to 1.5 X A ml., designated as
dilutions 1 and 1.5, respectively.
A. Measurements xvilh Glycerophosphate as Substrate
According to the principles of the Bodansky method, 3 plasma or eytolysate
was brought to act upon ^-glycerophosphate, using veronal buffer17 of pH 8.9 to
maintain the optimal alkaline reaction, and veronal-acetate buffers18 to study
the acid phosphatase activity at pH 4.9.
* Received for publication, November 22,194S.
167
168
MEHRENDT
E x a m p l e : 0.4 ml. plasma or cytolysate
0.5 ml. 1/20 M disodium glycerophosphate
2.0 ml. 5 per cent monosodium diethylbarbiturate
2.1 ml. distilled water.
These blood-substrate-buffer mixtures were incubated a t 37 C. for one hour. After addition of 3 ml. of 20 per cent trichloracetic acid and subsequent filtration, phosphorus was
determined in an aliquot of the clear filtrate according to t h e method of Fiske and SubbiiRow. 6 A control was set up and run in the same way, except t h a t the trichloracetic acid
was added prior to t h e incubation. T h e difference between t h e phosphorus content of
sample and control represents the phosphatase activity of the blood derivative. The
results are expressed as units of phosphatase activity, one unit being equivalent to one
TABLE 1
P H O S P H A T A S E ACTIVITY OF H U M A N PLASMA AND L A K E D EKYTHROCYTES, USING
SUBSTRATE ^ - G L Y C E R O P H O S P H A T E AND B U F F E R S V E R O N A L AT P H 8.9 AND
V E R O N A L - A C E T A T B AT P H 6.1 AND 4.9
EXPERIMENT
NO.
OF BLOOD
DONORS
ANTICOAGULANT
HEMATOCRIT,
PER CENT
52
49 yr.
Oxalate
40
53
32 yr.
Oxalate
41
63
64"
13 mo.
IS mo.
Heparin
Heparin
39
25
65 b
IS mo.
Heparin
21
73ac
6 mo.
Heparin
33
74a d
47 yr.
Oxalate
44
Diagnosis: " sickle cell anemia,
b
pH OF
BUFFERSUBSTRATE
S.9
4.9
S.9
4.9
8.9
S.9
4.9
8.9
6.1
4.9
S.9
4.9
S.9
4.9
c
PHOSPHATASE ACTIVITY. UNITS
In Plasma
In Cytolysate
In Erythrocytes
1.1
0.2
7.1
0.3
4.S
2.7
0.1
0.9
0.6
0.1
19.6
4.0
11.S
0.3
0.32
0.67
0.0
1.0
0
0.2
0.7
1.0
1.1
3.9
0.6
0.9
0
0.9
1.2
2.5
2.2
3.7
toxic a n e m i a , biliary obstruction,
d
—
1.2
4.2
7.1
7.7
27.9
2.7
4.1
—
3.0
P a g e t ' s disease.
milligram of phosphorus liberated per 100 ml. of plasma or cytolysate during one hour of
incubation. T h e phosphatase activity of erythrocytes was calculated as follows:
P h o s p h a t a s e units in erythrocytes = phosphatase units in cytolysate X
\f
where D = dilution of cytolysate
V = relative volume of red cells in whole blood.
As shown in Table 1, phosphatase activity is always present at pH 4.9, both
in plasma and cytolysate. In this acid medium, cytolysates at all times exhibit
greater activity than plasma. At pH 8.9, however, cytolysates show little, if
any, activity. Figure 1 summarizes the results obtained on normal human
blood (No. 52, 53, 63 of Table 1). On the whole, the results of these experiments
with glycerophosphate as substrate are not very impressive as far as the enzymic
action of cytolysates is concerned. The amount of phosphorus liberated from
the substrate by the acid and the alkaline phosphatases of cytolysates represents
169
PHOSPHATASES IN BLOOD
only fractions of the phosphorus concentrations liberated by the alkaline plasma
phosphatase. The outcome of experiments using phenylphosphate, however, is
more enlightening.
TABLE 2
P H O S P H A T A S E ACTIVITY O F H U M A N PLASMA AND L A K E D E R Y T H R O C Y T E S I N B L O O D O F
N O R M A L COMPOSITION, U S I N G SUBSTRATE SODIUM P H E N Y L P H O S P H A T E AND
B U F F E R V E R O N A L - A C E T A T E , D I L U T I O N FACTOR OF CYTOLYSATES 1.5
AND H E P A R I N AS ANTICOAGULANT
NO.
07
AGE OF
BLOOD DONOR
IN YEARS
2
HEMATOCRIT,
PER CENT
38
6S
6
3S
71
49
41
72
12
38
70
2
39
7S
3
38
79
S2
49
5
43
40
p H OF
BUFFERSUBSTRATE
8.9
0.1
4.9
S.9
6.1
4.9
3.8
8.9
6.1
4.9
3.8
8.9
7.6
6.1
4.9
3.8
2.6
8.9
6.1
4.9
S.9
4.9
4.9
S.9
4.9
PHOSPHATASE ACTIVITY UNITS
In Plasma
In Cytolysate
In Erythrocytes
9.2
0.4
0.6
6.3
3.3
3.6
2.S
5.3
2.4
2.4
3.1
5.0
0.2
6.7
23.9
1.2
16.7
21.7
14.8
1.3
21.0
26.0
15.4
1.0
3.1
21.5
29.0
24.0
9.2
1.3
58.1
6S.7
0.5
77.3
53.3
0
50.9"
26.5
94.4
4.7
66.0
S5.7
5S.5
4.S
76.9
95.1
56.4
4.0
12.2
85.0
114.6
94.S
36.3
5.0
223.7
264.5
2.0
305.3
1S5.9
0
127.3
—
2.1
—
2.1
—
10.3
—
9.9
S.S
7.3
0.7
11.S
5.3
o.s
" Dilution factor 1.0.
B. Measurements with Phenylphosphate as Substrate
Plasma or cytolysate obtained as described above was added to the buffered
substrate consisting of 1/200 M sodium phenylphosphate and Michaelis' veronalacetate buffers18.
E x a m p l e : 0.5 ml. plasma or cytolysate
5.0 ml. buffer
4.5 ml. sodium phenylphosphate.
The mixture was incubated a t 37 C. for one hour, 4.5 ml. of diluted phenol reagent was
added, and t h e phenol liberated was determined by the method of G u t m a n and G u t m a n . 8
A control was treated similarly, except t h a t the phenol reagent was added prior to in-
170
BEIIRENDT
cubation. T h e difference in t h e a m o u n t of phenol content between t e s t sample a n d control gave the phosphatase activity of the sample. The results were expressed as units of
phosphatase activity, one unit being equivalent to t h a t amount of enzyme which will
liberate 1 mg. of phenol per 100 ml. of blood derivative from t h e phcnylphosphate s u b s t r a t e
during one hour. T h e phosphatase content (in units) of the erythrocytes was calculated as
described above.
The results of 13 such experiments are given in Tables 2 and 3. In principle,
they confirm the findings obtained with glycerophosphate, but make the action
of the acid enzyme appear more definite. When the activity of cytolysates, as
measured in these experiments, is expressed in units of red cell phosphatase and
compared with the amount of plasma phosphatase units from the same blood
TABLE 3
P H O S P H A T A S E ACTIVITY OP H U M A N P L A S M A AND L A K E D ERYTHROCYTES I N BLOOD
OF ABNORMAL C O M P O S I T I O N , U S I N G SAME S U B S T R A T E , B U F F E R S , D I L U T I O N
FACTOR OF CYTOLYSATES, AND ANTICOAGULANT AS I N T A B L E 2
EXPERIMENT
NO.
73b
74b
75
77
S3
DIAGNOSIS
Biliary obstruction
Paget's disease
AGE OF
BLOOD
DONOR
6 mo.
47 yr.
Biliary obstruction
12 yr.
Prostatic cancer
with metastases
56 yr.
Sarcoma of femur
10 yr.
HEMATOCRIT,
P E R CENT
33
44
32
40
39
p H OF
BUFFERSUBSTRATE
S.9
4.9
8.9
6.1
4.9
8.9
6.1
4.9
8.9
6.1
4.9
8.9
4.9
PHOSPHATASE ACTIVITY, UNITS
In Plasma
28.1
9.4
12.9
—
7.7
45.5
—
5.3
5.4
—
20.6
34.5
7.8
In Cytolysate
In Erythrocytes
1.2
41.5
2.9
36.2
58.2
9.5
64.7
78.2
5.5
188.6
10.0
123.4
198.5
44.5
303.3
366.6
—
—
51.S
66.2
1.1
49.6
194.3
248.3
1.4
190. S
sample, the following relationship appears to be typical for human blood of any
age group and of normal composition (Table 2 and Fig. 2): Phosphatase activity
at pH 8.9 is more than twice as great in the plasma as in the red cells, whereas
at pH 4.9 erythrocytes possess an activity at least a hundredfold that of plasma.
The values of acid red cell phosphatase range between 94.4 and 305.3 units, with
an average of 159.1, while King and his collaborators15 found somewhat higher
figures, namely, a range from 200 to 400 units, with an average of 340.
A second group of determinations (Table 3) was performed on blood samples
from patients whose diseases affected the relative red cell volume or the activity
of alkaline or acid plasma phosphatase. In these abnormal specimens, the acid
phosphatase of the red cells was found to be higher than in cells of normal blood,
averaging 238.4 units, with a range between 188.6 and 366.6 units. The number
of estimations in each group of diseases is too small to permit any conclusions
as to the significance of such an apparent increase.
171
PHOSPHATASES IN BLOOD
CYTOLYSATES, RED CELL SUSPENSIONS, AND WHOLE BLOOD
An occasional experiment revealed that the hydrolytic effect of whole blood
upon buffered phenylphosphate was almost as strong as that of laked erythrocytes
when the blood was prevented from being hemolyzed during the incubation
period. This observation prompted the investigation of the phosphatase
activity of nonhemolyzed red cells under varying conditions.
T o insure preservation of their physical integrity during the period of enzymic action,
erythrocytes were brought to act upon the substrate as follows: X ml. of heparinized
blood were centrifugcd and t h e plasma removed. T h e remaining cells were washed 3 t i m e s
TABLE 4
P H O S P H A T A S E ACTIVITY O F L A K E D AND S U S P E N D E D H U M A N ERYTHROCYTES I N B L O O D
OF N O R M A L COMPOSITION U S I N G S U B S T R A T E ISOTONIC SODIUM P H E N Y L P H O S P H A T E , AND B U F F E R ISOTONIC V E R O N A L - A C E T A T E - S O D I U M C H L O R I D E
AT P I I
4.9
PHOSPHATASE ACTIVITY, UNITS
HEMATOCRIT,
PER CENT
EXPERIMENT N O .
In Cytolysate
In Suspension
In Erythrocytes
Dilution
Calculated from
Dilution 1.5
1.0
92
99
71
72
83"
84
85
91
94
95
96"
Diagnosis:
41
42
41
38
39
43
41
42
41
41
34
a
1.5
—
—
43.8
26.0
29.0
49.6
12.5
11.4
13.6
25.7
72.9
35.7
23.8
34.0
38.4
45.3
30.3
44.5
24.0
60.7
28.4
3.0
Cytolysate
26.3
—
95.1
114.6
190.8
43.6
40.7
48.6
94.0
266.7
157.5
Suspension
160.2
187.9
105.4
134.2
147.7
105.4
110.9
106.0
87.S
246.5
125.3
sarcoma of femur, b sickle cell anemia.
with physiologic saline solution and then resuspended in enough saline to bring t h e volume
of the suspension u p t o t h e original sample volume of X ml., or t o 1.5 times X m l . , or t o 3
times X ml. T h e dilutions were designated as 1, 1.5, and 3, respectively. I n portions of
these suspensions, the phosphatase was estimated under isotonic conditions, using an
isotonic substrate-buffer mixture of p H 4.9 consisting of 0.5 p a r t s 0.107 M (2.34 per cent)
disodium phenylphosphate, and 9.5 p a r t s of Michaelis' isotonic veronal-acetate buffers. 18
A hematocrit determination was made on a separate blood sample.
E x a m p l e : 0.5 ml. blood derivative (red cell suspension)
0.5 nil. isotonic phenylphosphate
9.5 ml. isotonic buffer solution.
T h e reaction mixture was almost always free of hemolysis a t the end of the incubation
period, giving proof of t h e isotonicity of t h e set-up. Mixtures showing traces of hemolysis
were discarded. The incubation and colorimetric determination of the liberated phenol
and the calculation of phosphatase activity as units were carried out as described above for
t h e cytolysates. F o r the comparative s t u d y of cytolysates, t h e same isotonic s u b s t r a t e buffer mixture was employed as for the red cell suspensions.
172
BEHRENDT
After 2 preliminary measurements (No. 92 and 99 in Table 4) had shown that
red cell suspensions exert a strong enzymic activity upon phenylphosphate,
comparative determinations were made on cytolysates and suspensions derived
from the same blood sample. The results are recorded in Table 4. -As may be
seen from the 2 last columns of this table, in 8 of 9 cases the erythrocyte suspensions exerted phosphatase activity at least as strong as that of the hemolyzed
cells. In one experiment (No. 83), the activity of the suspensions was slightly
less than that of the cytolysate, but in 6 instances (No. 71, 72, 84, 91, 96) the
action of the suspension was superior to that of the cytolysate.
If the outcome of these tests may be taken as sound evidence, it would appear
that the phosphatase principle, when retained within the red cell membrane,
produces an equal or even stronger enzymic action upon the extracellular substrate than the same enzyme when released by cytolysis into the substrate-buffer
TABLE 5
P H O S P H A T A S E ACTIVITY OP H U M A N ERYTHROCYTES S U S P E N D E D I N V A R I O U S M E D I U M S
I N BLOOD OP N O R M A L C O M P O S I T I O N U S I N G SUBSTRATE ISOTONIC SODIUM
P H E N Y L P H O S P H A T E , B U F F E R ISOTONIC V E R O N A L - A C E T A T E - S O D I U M
C H L O R I D E AT P I I 4.9 AND ANTICOAGULANT H E P A R I N
PHOSPHATASE ACTIVITY, UNITS
EXPERIMENT
NO.
In Plasma
In Whole
Blood
In Cell Suspension in
Saline, Dilution
Factor 1.0
In Cell Suspension -f Plasma, in Proportion
1:1
1:2
1:3
—
—
—
—
—
84
1.0
22.S
45.3
85
1.0
11.6
45.5
92
—
—
65.7
4.5
—
—
—
94
0.5
27.2
36.0
27.9
1.7
—
95
1.9
61.5
91.1
60.0
96"
3.3
35.9
42.6
40.5
0
° D i a g n o s i s , sickle cell a n e m i a .
medium. Some further evidence may be of help in explaining this finding. No
phosphatase activity could be ascertained in pooled samples of the saline solutions used for washing the red cells. Negative results were also obtained with
the supernatant liquid of a mixture consisting of 0.5 ml. of red cell suspension
and 9.5 ml. of isotonic buffer solution, when tested for phosphatase after incubation for one hour.* I t may be concluded that neither during the process of
washing: nor during the period of incubation does the active principle penetrate
through the red cell membrane into the extracellular phase of the suspension.
W H O L E BLOOD AND E R Y T H R O C Y T E
SUSPENSIONS
As a result of these findings, it seemed desirable to test the phosphatase
activity of whole blood, i.e., of red cells suspended in plasma. Employing the
same isotonic arrangement as before, comparative estimations were made on
* T h e s e t e s t s were m a d e upon t h e suggestion of D r . L. Michaelis of New Y o r k .
173
PHOSPHATASES IN BLOOD
plasma, whole blood, and erythrocytes suspended in saline solution, all prepared
from the same blood specimen. The findings are listed in Table 5. When
tested at pH 4.9, whole blood was found to be consistently less active than the
red cell suspension containing a similar (percentile) volume of erythrocytes.
TABLE 6
E F F E C T OF PLASMA ON THE P H O S P H A T A S E ACTIVITY O F R E D C E L L CYTOLYSATES IN
N O R M A L H U M A N B L O O D , U S I N G SUBSTRATE SODIUM P H E N Y L P H O S P H A T E AND
'
B U F F E R V E R O N A L - A C E T A T E AT P H 4.9
PHOSPHATASE ACTIVITY, UNITS
EXPERIMENT NO.
Cytolysate + Plasma, in Proportion
Cytolysate, Dilution 1.0
38.6
109.4
S3.9
94
95
97
1:1
1:2
4.1
30.5
19.5
2.1
160
140
r
4 -
«. 120
-^
'B
=
.100
p
LU
CO
£
I
85 60
o
£ 40
3 2 -
20
E
1 -
0I
80
<£.
~~
At pH 8.9
1 1
At pH 4.9
FIG.
1
0
At pH 8.9
FIG.
At pH 4.9
2
F I G . 1. Units of phosphatase a c t i v i t y of human plasma (P) and erythrocytes (E) in substrate/3-glycrophosphate. Values represent averages of experiments No. 52, 53 and 63 from Table 1.
F I G . 2. Units of phosphativse activity of human plasma (P) a n d erythrocytes (E) in s u b s t r a t e phenylhosphatc. Values represent averages from Table 3.
In other words, the presence of plasma in the medium surrounding the intact
cells seems to interfere with the enzymic action of the intracellular phosphatase
upon the phenylphosphate in the extracellular medium.
If this assumption is correct, the activity of red cells suspended in saline solution will be reduced by the addition of plasma at the start of the incubation
period. This was tested in four experiments.
174
BEHRENDT
Example: 0.25 ml. red cell suspension
0.5 ml. isotonic phenylphosphate
9.5 ml. isotonic veronal-acetate buffer
+0.25ml. plasma.
Table 5 reveals that addition of plasma did inhibit the phosphatase activity of
cell suspensions in all instances. When the proportion of plasma to suspension
was 1:1, phosphatase activity was reduced to approximately that of whole blood
in 3 of the 4 tests. When the ratio of plasma to suspension was further increased (2:1 and 3:1) the phosphatase activity of the suspended erythrocytes
declined sharply or disappeared completely. The actual concentration of
plasma in the incubated mixture varied from 2.4 to 6.8 per cent in the different
tests.
This inhibitory action of plasma, however, can also be demonstrated on solutions of laked red cells tested in the non-isotonic set-up described above. Table
6 shows that the inhibition by plasma of the phosphatase contained in cytolysates
was strong or almost complete, depending upon the ratio of plasma to cytolysate
in the substrate-buffer mixture.
COMMENT AND SUMMARY
The following acid phosphatases have been found in human blood since Folley
and Kay 7 presented their classification of the various phosphatases:
(1). The serum acid phosphatase occurring in normal blood, with an optimal
activity at pH 4.9-5.0.9 Its action is markedly inhibited by sodium fluoride
and citrate, and not accelerated by magnesium chloride. This enzyme splits
a-glycerophosphate with no greater speed than /3-glycerophosphate, while the
scission of phenylphosphate is most easily catalyzed. These properties coincide
with those of the acid phosphatases present in liver, spleen, kidney and bone,
organs assumed to be the source of this normal serum acid phosphatase. 21
(2). The serum acid phosphatase appearing in the plasma of patients with
metastasizing cancer of the prostate. 1,10 Its source is the prostatic cancerous
tissue.11 In some respects, the enzyme resembles the normal serum acid phosphatase 2 : optimal activity at pH 4.9 and inhibition by fluoride; it is, however, also
inhibited by alcohols, for instance ethyl alcohol,13,21 and is little affected by
citrate. 21
(3). The erythrocyte acid phosphatase, with an optimal pH at 4.9, present
in cells of persons of all ages. 2,15 While this enzyme, according to some observers,2, 12 is not significantly affected by fluoride or magnesium, an inhibition
by both ions has been found by others.15 The action of this enzyme upon phenylphosphate is more than 100 times greater than that of the normal serum acid
phosphatase. The phosphatases occurring in the erythrocytes of horse, rabbit,
and guinea pig differ from the acid phosphatase of red cells of man in various
respects, chiefly in their optimal pH. 1 9 , 2 0
While the properties of human red cell phosphatases and the kinetics of the
reactions they catalyze, have so far been investigated on laked whole blood or
laked erythrocytes, the present report includes a study of the intracellular
PHOSPHATASES IN BLOOD
175
phosphatase in living cells. By using an isotonic set-up of cell suspensions,
buffer and substrate, an attempt has been made to study in vitro the factors
governing the substrate-enzyme accessibility in vivo. From these first observations, it would seem that at pH 4.9 phenylphosphate must penetrate through
the erythrocyte membrane in order to be acted upon by the enzyme. This
would resemble the penetration of such permeants as phenylacetate, i.e., the
introduction of a phenyl in the place of a hydrogen, favoring the lipoid solubility
of the permeant. However, unlike phenylacetate, phenylphosphate has no
hemolytic effect, probably because of its ionization.* It should not be difficult
to supply experimental proof of such a permeation. The measurement of
anhydrase activity within living, suspended red blood cells by Booth4 and by
Keilin and Mann11 confirms the potential significance of quantitative enzyme
studies on suspended red corpuscles. The isotonic arrangement would also
permit comparison of the kinetics of phosphatase reactions catalyzed by intact
living erythrocytes and by red cell cytolysates. Obviously, the discrepancy
between fermentation by yeast cells and by yeast extracts 6,16 invites speculation
on such a comparison. However, final conclusions as to the activity of "cytolysates" as employed in the present studies should await the results of other
experiments in which the activity of leukocytes and platelets has been properly
taken into account.
CONCLUSIONS
Our observations indicate that at pH 4.9, phenylphosphate must penetrate
through the erythrocyte membrane in order to be acted upon by the enzyme,
phosphatase.
Acid phosphatase activity of red cells is reduced in the presence of plasma.
This inhibition is equally strong when suspensions or cytolysates are employed.
Apparently, the action of plasma upon the enzyme is not mediated by osmotic
factors bound to the presence of intact cells.
REFERENCES
1. BAHRINGER, B . S., AND WOODAHD, H . Q . : P r o s t a t i c carcinoma with extensive i n t r a prostatic calcification, with discussion of possible role to prostatic phosphatase.
T r . Am. A. Gonito-Urin. Surgeons, 31: 363-369,1938.
2. B E H R E N D T , H . : Phosphatase activity of human erythrocytes. P r o c . Soc. Exper.
Biol, and Med., 54: 268-270,1943.
3. BODANSKY, A.: Notes on determination of serum inorganic phosphate and of serum
phosphatase activity. Am. J . Clin. P a t h . , Tech. Suppl., 7: 51-59,1937.
4. BOOTH, V. H . : Carbonic anhydrase activity inside corpuscles. Enzyme-substrate
accessibility factors. J . Physiol., 93: 117-128, 1938.
5. BROOKS, S. C.: Permeability and enzyme reactions. I n : Advances in Enzymology.
Vol. 7, New Y o r k : Interscience, p p . 1-34, 1947.
6. F I S K E , C. H . , AND SUBBAROW, V.: T h e colorimetric determination of phosphorus.
J. Biol. Chem., 66: 375-400, 1925.
7. FOIJJBY, S. J., AND K A Y , H . D.: T h e phosphatases. Ergebn. d. Euzymforsch., 6:
159-212, 1936.
S. CUTIMAN, A. B . , AND GuTMAN, E . B . : " A c i d " phosphatase activity of serum of normal
human subjects. Proc. Soc. Exper. Biol, and Med., 38: 470-473, 1938.
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