THE SURFACE CHEMISTRY OF BONE*

III. OBSERVATIONS ON THE ROLE OF PHOSPHATASE

BY W. F. NEUMAN, V. DGTEFANO, AND B. J. MULRYAN

(From the Division of Pharmacology and Toxicology, Department of Radiation

Biology, School of Medicine and Dentistry, The University of Rochester,
Rochester, New York)

(Received for publication, May 19, 1951)

The enzyme phosphatase has been mentioned in almost every scheme of

calcification since Robison (1) first suggested that this enzyme provided
local, high concentrations of inorganic phosphate by virtue of its action
on a suitable substrate. Perhaps the principal reason for the persistence
of this idea is that an almost perfect correlation has been repeatedly ob-
served (2-7) between the histological sites of phosphatase activity and the
areas of active calcification.
Robison, however, found that ester phosphate was present only in low
concentrations in plasma (8). In an attempt to determine the means by
which phosphatase is supplied with suitable substrate, recent workers have
focused attention on the importance of glycogen and the phosphorylative
cycles of glycolysis in calcification (g-12). These investigators have shown
the glycolytic processesto be important in calcification, but they have not
demonstrated that the phosphorylative reactions involved furnish substrate
for phosphatase. This may be illustrated by quoting from Gutman and
Yu (13): “Whether glycogenolysis is essential in order to provide phos-
phorus in some special form or concentration or place, or whether required
energy is made available, is not clear.”
At the moment, the phosphatase question appears to involve two con-
flicting pieces of information: (a) the enzyme is invariably found to be
present in areas of active calcification, suggesting a key r&e in the deposi-
tion of osseousmineral, and (b) no studies yet conducted have provided
adequate evidence that the enzyme, though present, has sufficient sub-
strate in vivo to exert a significant effect on the concentration of inorganic
phosphate.
If one assumesthat ester phosphate is inhibitory to calcification, the
problem is partially resolved. Provided that ester phosphate is sufficiently
inhibitory, a high concentration of substrate is not required for phosphatase
* This paper is based on work performed under contract with the United States
Atomic Energy Commission at The University of Rochester Atomic Energy Proj-
ect, Rochester, New York.

This is an Open Access article under the CC BY license.

228 SURFACE CHEMISTRY OF BONE. III

to play an important r6le in calcification. The enzyme may be acting as

a trigger mechanism by removing an agent which prevents the precipita-
tion of calcium phosphate salts.
Reported below are the results of a series of experiments designed to
test this hypothesis.

EXPERIMENTAL

Methods
The preparation of ashed bone specimens, analytical methods, and radio-
activity procedures were the same as those reported previously (14).

TIME IN DAYS
FIG. 1. The inhibition of mineral accretion in vitro by the presence of glycero-
phosphate, 10 mg. per cent of ester P. 20 mg. specimens of fresh, defatted veal
bone were placed in 10 ml. samples of a synthetic ultrafiltrate having the same com-
position as the basic solution (see the text). After 24 to 72 hours agitation at 37,
the suspension was centrifuged, the solution aspirated for analysis, and fresh solu-
tion added. In the graph are plotted the amounts of inorganic phosphate removed
from the solution by the bone preparation.

Results
The first experiments were performed only to show grosseffects. It had
been previously demonstrated (15) that powdered bone preparations un-
dergo mineral accretion when immersed daily in a synthetic ultrafiltrate
of plasma. This solution, by itself, is stable for months; no precipitation
occurs in the absence of bone. These experiments were repeated. In
addition, a representative phosphate ester, a-glycerophosphate, was added
to the ultrafiltrate. The results are given in Fig. 1. Clearly, the presence
of ester inhibited the formation of a new solid phase.
The above experiments were repeated with one modification: the ester
was added as the calcium salt. For the most part, the additional calcium
prevented the inhibitory action of ester phosphate as is seen in Table I.
It was of interest to test whether ester phosphate forms soluble, undis-
sociated complexes with calcium, thereby inhibiting the precipitation of
insoluble calcium salts. Many of the common techniques used in com-
plex ion studies such as polarography, spectrophotometry, electrochemistry,
 W. F. NEUMAN, V. DISTEFANO, AND B. J. MULRYAN 229

etc., are not readily applicable to an investigation involving calcium.

Therefore, the effect of ester phosphate on the solubility of calcium sulfate
was examined. If complex formation occurred, the reduction in free cal-
cium ion activity would result in a reduced amount of solid calcium sulfate
given by a fixed concentration of sulfate ions.
TABLE I
Ej’ect of Added Calcium Glycerophosphate on Average Daily Mineral Accretion of Bone
in Inorganic System

Fresh bone Ashed bone

Constituent
Constituent added analyzed
~~~t~i
Control Control %Yt%
___-
mg. per day mg. per day mg. $cr day mg. per day
Free ester.................. 0.65 0.22 0.81 0.33 Phosphate
“ “ “
0.51 0.20 0.63 0.24
Calcium salt of ester. 0.72 1.13 1.11 1.60 Calcium
“ I‘ “ “ 0.38 0.41 0.88 0.71 Phosphate

The procedures used were the same as those given in the legend to Fig. 1.

0.0.
*

3 0.6. F”
0” G+ A
04- ’
B 0.2. l /

2
2 ,/f
,/j., . , , , I.,

d
d 3 4 5 6 7 8 9 IO II I2
12 13
(3 MILLIMOLES CALCIUM ADDED
2. The effect of added glycerophosphate
FIG. on the solubility of calcium sulfate.
0, no ester added, 2 days equilibration; A, no ester added, 2 weeks equilibration;
A, ester added, 2 weeks equilibration; 0, ester added, 5 weeks equilibration. 0,
ester added 2nd day after precipitation, 7 weeks equilibration.

Varying amounts of CaC12 solution were added to a solution containing

15 mM of sodium sulfate, both in the presence and absence of 2 mM of
sodium cr-glycerophosphate. The mixtures were adjusted to a total volume
of 100 ml. and set aside for varying periods of time at room temperature.
The precipitated mixtures were then filtered through small, sintered, glass
filter sticks and the precipitates dried at 115” and weighed. The results
are presented in Fig. 2.
The principal effect of ester was to increase the time required for pre-
230 SURFACE CHEMISTRY OF BONE. III

cipitation. When calcium sulfate was allowed to precipitate before the

addition of ester, only small solubilizing effects were noted. These re-
sults indicate that the action of complex formation of ester on calcium ion
is almost negligible. Perhaps ester phosphate delays precipitation by
“poisoning” the nuclei on which aggregation takes place in the manner
reported for other anions (16).
If such a “poisoning” action were operative, it should be possible to
demonstrate the presence of ester phosphate in the solid phase. Analyses
of washed calcium sulfate precipitates formed in the presence of ester phos-
phate did indeed show the presence of small but detectable amounts of

LOG -$

MG. ESTER
I G. BONE

0.11
001 01 IO IO
LOG C
(FINAL CONCENTRATION MG% ESTER P)
FIQ. 3. The adsorption of o-glycerophosphate by powdered bone ash. 20 mg.
specimens of glycol-ashed veal bone were shaken in 10 ml. portions of 0.025 M so-
dium bicarbonate (previously equilibrated with 1 atmosphere of 5 per cent CO*)
containing radioactive a-glycerophosphate for 29 hours at 37”. The suspensions
were then centrifuged and the supernatant solution taken for radioactivity
measurement.

ester phosphate. These results, however, could be attributed to contami-

nation. Accordingly, a more direct test was devised.
a-Glycerophosphate containing radioactive P32 was prepared (17) and
carefully purified by the repeated addition and removal of carrier inert
phosphate, to insure the absence of contaminating inorganic radiophos-
phate. Solutions containing varying amounts of this ester at physiolog-
ical pII were shaken with powdered glycol-ashed veal bone for 29 hours.
Preliminary experimentation established two facts: (a) equilibrium was
reached in less than 24 hours, and (b) the radioactive phosphate present
in the ester did not undergo exchange with inorganic phosphate in solution.
The bone specimenswere separated from the solutions by centrifugation
and the amount of ester adsorbed was determined from the amount of
radioactivity taken up by the bone. These results are presented in Fig. 3.
 TV. F. NEUMAN, V. DISTEFANO, AND B. J. MULRYAN 231

The results were satisfactorily described by a Freundlich isotherm (18)

and indicate that, at physiological concentrations of ester, 0.1 to 1 mg.
per cent of P (8), the ester is strongly adsorbed by bone.
The experiments reported above dealt only with isolated, inorganic
systems and do not permit generalization. As a more practical approach
to the problem, the effect of ester phosphate on the calcification of rachitic
cartilage slices in vitro was undertaken.
The experimental procedure was a slightly modified version of that first
described by Robison et al. (19). The degree of calcification of the incu-
bated slices was estimated by the scoring method of Sobel et al. (20).
Weanling, female, albino rats of the Wistar strain, 22 days of age, were
maintained on a standard U. S. P. rachitogenic diet (Nutritional Biochem-
icals Corporation, No. 2) for a period of 26 days, after which they were
sacrificed with chloroform. Two slices of approximately the same size and
thickness were cut by free-hand section from the epiphyseal cartilage of
each tibia. The four slices obtained from each animal in this manner
comprised a series. One rachitic slice of each series was placed in a small
Erlenmeyer flask containing 50 ml. of one of the four incubating solutions.
0.4 ml. of 0.01 per cent phenol red solution was added to each incubating
mixture (to detect gross changes in pH), and the whole was equilibrated
with 5 per cent CO, in 95 per cent Nz at atmospheric pressure. The
flasks were then stoppered, sealed with tape, and agitated for 18 hours in
a constant temperature room. The rachitic sections were then removed,
washed with distilled water, and stained with 5 per cent silver nitrate, and
the degree of calcification was evaluated by inspection. The composition
of the four incubating solutions was as follows: Solution CP, the basic
solution, had the following molar composition: sodium 0.115, bicarbonate
0.022, chloride 0.094, calcium 0.0031, inorganic phosphate 0.0016, mag-
nesium 0.0006, and citrate 0.0004. Solution CPE, the ester control, had
the same composition as Solution CP except for the presence of a-glycer-
ophosphate (0.125 per cent P) added as the dicalcium salt. Solution CPI,
the mercury control, was the same as Solution CP except for the presence
of Hg as 0.001 M HgC12. Solution CPIE, the experimental solution, was
the same as Solution CP except for the presence of both 0.001 M HgCl,
and 0.125 per cent P as dicalcium a-glycerophosphate.
The results are given in Table II. A comparison of the results with
Solutions CP and CPE shows that the addition of small quantities of ester
phosphate enhanced calcification in normal sections (active phosphatase
present). Comparison of Solutions CP and CPI illustrates the strong in-
hibition of calcification by the presence of mercury. Whether this inhi-
bition can be attributed entirely to the action of mercury on phosphatase
is doubtful (21). None the less, the addition of ester phosphate to such
232 SITRFACE CHEMISTRY OF BONE. III

mercury-poisoned sections reduced even further the degree of calcification

observed (Solution CPIE versus Solution CPI). In other words, ester
TABLE II
Effect of Added Calcium Glycerophosphate on Calcification* of Normal and
Phosphatase-Inhibited Cartilage Slices from Rachitic Rat Tibia

Incubating solution
Rat No.
CP CPE CPI CPIE
I I I

Temperature 37”

1 24 (++++I 3 c++++j 1 (++++I 1 (++)

2 2 c++++j 21 (++++I 1 (+I 0
3 2 c++++j 24 c++++j 1; (++++I 1 (++)
4 at t++++j 21 (++++) 13 (++++) 1 (+I
5 2 c++++j 2 c++++j 1 (++) 0
6 2 (++++I 2 (++++I 1 (+++) 1 (+)
7 2 (++++I 3 c++++j 1 (++) 0
8 2 c++++j 2) c++++j 2 (++I 3 (++++I
9 2 (++++I 2 c++++j 1 (+++) 1 (++)
10 3 c++++j 33 c++++j 2 (++++I 1 (++++I
11 3 c++++j 4 c++++j 2 (++++I 1 c++++j
12 3 (++++I 3 (++++I 2 c++++j 1 (++++I

Temperature 1”

13 1t (++++) 2 (++++I 1 (+I 0

14 13 (++++I 2 t++++j 1 (+) 0
15 la c++++j 2 (++++) 1 (+) 0
16 23 c++++j 24 (++++I 0 0
17 2 (++++I 2% (++++I 1 (+I 0
18 2? c++++j 23 (++++I 1 (+I 0
19 13 (++++) 2 (++++I 1 (+) 0
20 2 (++++I 2 (++++I 1 (++I 1
21 la (++++) 2 (++++I 1 (++I 0
22 2 (++++I 2 (++++I 1 (+I 0
23 2 c++++j 2 c++++j 1 (++++I 0
24 2 (++++I 2 c++++j 1 (+++I 1 c+++j

* The degree of calcification was scored according to the method of Sobel et al.
(20): 0 to 4 in the longitudinal direction and 0 to ++++ in the transverse direc-
tion. The composition of the various incubating solutions and procedures are given
in the text.

phosphate was beneficial in sections containing active phosphatase but

harmful in those containing inactivated phosphatase.
While the results given in Table II were reasonably consistent, occasional
aberrant values were obtained. An experiment was performed to deter-
 W. F. NEUMAN, V. DISTEFANO, AND B. J. MULRYAN 233

mine whether this variability could be attributed to occasional incomplete

inactivation of phosphatase by mercury. One group of slices of rachitic
tibia served as a control and was placed in isotonic saline for 2 hours.
A second group of slices was placed in isotonic saline containing 1 m.eq.
of Hg++ per liter as HgCl, for 2 hours. All slices were then washed in
saline and placed in individual 5 ml. portions of 0.001 M phenolphthalein
phosphate solution in Verona1 buffer for 2 hours. Immediately after the
addition of 5 ml. of glycine buffer, the color intensity, which is proportional
to the phosphatase activity, was determined by means of a calorimeter.
The reagents were those described by Huggins and Talalay (22). The
TABLE III
Inconsistent Inhibition of Phosphatase in Rachitic Cartilage Slices by Mewury

Phosohatase activity*
Animal No. -
Control Hg-treated
I

1 ! 2.6
” 23 -
3”
“._

5.4
4 2.9 1.5
5 2.7 -
6 2.8 1.1
7 3.6 -
8 2.2 -
9 3.6 1.0
10 3.6 -
11 6.2

* Activity is expressed as mg. of phenolphthalein phosphate liberated per liter

per hour. Values less than 1 mg. are of doubtful significance and are indicated by
a dash.

results are given in Table III and indicate that mercury consistently in-
hibited phosphatase activity, but did not completely inactivate the en-
zyme in all cases.

DISCUSSION

The results reported above clearly show that ester phosphate is strongly
adsorbed by bone ash. Further, the presence of such adsorbed ester
inhibits the accretion of additional mineral both in isolated inorganic
systems and in calcifying slices of rachitic cartilage taken from rat tibia.
In the latter instance, these effects were noted with very low physiological
concentrations of ester, 0.125 mg. per cent of ester P.
Since ester phosphate showed little ability for complex formation with
calcium ions in solution, the mechanism seems to involve the interference
234 SURFACE CHEMISTRY OF BONE. III

of the adsorbed ester, with the subsequent addition of ions to preformed

crystal surfaces. This mechanism is not at all unreasonable. It seems
unlikely that a solid lattice structure such as that found in bone mineral
can be formed by the simultaneous collision of from 5 to 18 ions, all with
the correct energies. The stepwise addition of ions to a preformed sur-
face would be much more feasible. In such a case, the presence of an
adsorbed organic radical could easily block further addition of ions.
These data offer support for the hypothesis that at least part of the func-
tion of phosphatase in sites of active calcification is to remove the inhibit-
ing influence of ester phosphate at these sites. It is difficult, however, to
evaluate critically the importance of this phenomenon under the conditions
obtaining in vivo for two reasons: (a) The results obtained with the system
calcium sulfate-ester phosphate indicated that the process was merely a
“delayed” rate in the attainment of final equilibrium. W’hat this could
mean in terms of the dynamic conditions in vivo cannot be stated at present.
(b) These studies were performed with one representative ester, cu-glycero-
phosphate. It remains to be established whether all such physiological
phosphate esters behave similarly.

SUMMARY

Experiments were performed to test the notion that ester phosphate is

inhibitory to calcification in the absence of active phosphatase. It was
shown that ester phosphate is strongly adsorbed by bone mineral and that
such adsorbed ester inhibits the accretion of additional mineral both in
isolated inorganic systems and in calcifying slices of rachitic cartilage
taken from rat tibia. The significance of these results is discussed.

The authors gratefully acknowledge the assistance of M. W. Cucci in

the preparation of the radioactive a-glycerophosphate.

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