/. Embryol exp. Morph. Vol. 53, pp. 203-211, 1979
Printed in Great Britain © Company of Biologists Limited 1979
203
Phosphatase activity and ion levels associated with
normal and abnormal skeletogenesis in the fetal rat
By ELAINE S. BRUMMETT AND E. MARSHALL JOHNSON 1
From the Daniel Baugh Institute, Thomas Jefferson University,
Philadelphia
SUMMARY
Limbs from rat fetuses exposed to the alkyiating agent chlorambucil on day 14 of gestation
undergo abnormal skeletogenesis (Brummett & Johnson, 1979). The abnormal developmental
sequences are manifest as marked disruption of chondrogenesis and ossification. The present
study examines some of the biochemical factors normally related to initial mineralization and
their possible alteration during teratogenesis.
The specific activities of the acid, alkaline and pyro-phosphatases were determined for
normal and abnormal (drug-treated) fetal rat limbs during days 15-17 of gestation, the period
of cartilage-model calcification. This was done to determine if altered enzymatic activity
leading either to depressed localized phosphate levels or hydroxyapatite crystal growth
inhibition would account for the decreased calcification. The level of alkaline phosphatase
increased dramatically (sixfold in 3 days), in association with normal cartilage formation. In
the abnormal limbs, this rise was only half as great. Pyrophosphatase and acid phosphatase
activity in both normal and abnormal limbs did not change over the 3-day period.
The normal concentrations of ions critical to calcification, i.e. phosphate, calcium and
magnesium, were determined for maternal serum, amniotic fluid, whole fetuses and fetal
limbs. Mean phosphate and calcium levels increased slightly in the limbs with increasing
gestational age. The ion levels in drug-treated fetuses were within normal limits except in
amniotic fluid where phosphate was increased significantly on day 17. Also the protein
concentration in the fluid was elevated on day 16 and 17.
INTRODUCTION
Alkaline phosphatase has long been considered as having an active role in
the normal calcification of cartilage (Gomori, 1943) although its exact role is
unknown. Bonucci (1967) and Anderson (1969) have described extracellular
(matrix) vesicles that are morphologically related to initial calcium phosphate
crystal deposition in calcifying cartilage and alkaline phosphatase, pyrophosphatase and ATPase activity have been reported to be associated with
these vesicles (Ali, Sajera & Anderson, 1970; Salomon, 1974). It is thought
that an accumulation of phosphate occurs within the matrix vesicles through
the enzymic hydrolysis of oligo or polyphosphate compounds having a terminal
phosphate (Arsenis, Rudolph & Hackett, 1975). Precipitation of calcium
1
Author's address: Daniel Baugh Institute, Thomas Jefferson University, 1020 Locust
Street, Philadelphia, Pennsylvania 19107, U.S.A.
204
E. S. BRUMMETT AND E. M. JOHNSON
phosphate would then proceed either because of the increased quantity of
available phosphate or because known inhibitors of calcification such as
pyrophosphate, ADP and ATP (Fleisch & Russell, 1970) have been removed.
Some studies indicate that the enzyme activity of the matrix vesicles is due
to a single enzyme (Majeska & Wuthier, 1975; Felix & Fleisch, 1976) but
immunodiffusion and other studies indicate the presence of two enzymes
(Arsenis, Hackett & Huang, 1976). One of these enzymes was more active
toward linear oligophosphates including pyrophosphate so would be termed
inorganic pyrophosphatase and the other had activity on only simple phosphate
esters such as /7-nitrophenylphosphate, typical of alkaline phosphatase.
The present study examines /?-nitrophenyl- and pyro-phosphatases as pHdependent specific activities in fetal rat limbs during the 3 days when the
cartilage models of long bones are beginning calcification. Limbs from fetuses
exposed to the alkylating agent chlorambucil, and known to undergo abnormal
skeletogenesis (Brummett & Johnson, 1979), were compared to controls for
enzymic activity as well as ionic levels. A correlation was sought between the
activity of one or more phosphatases and the reduced calcification of drugtreated limbs in order to determine if local phosphate reduction, crystal formation inhibition, or excessive calcified matrix resorption were involved.
The normal concentrations of phosphate, calcium and magnesium were
measured in maternal serum, amniotic fluid, whole fetuses and limbs during
the stage of cartilage model mineralization. These values were compared to
those of abnormally developing limbs to determine whether or not ionic
imbalance or the reduced availability of one or more of these crucial ions
might play a role in abnormal calcification.
MATERIALS AND METHODS
Nulliparous Long-Evans rats weighing 180-240 g, diagnosed as proestrus,
were caged overnight with males of the same strain. The presence of sperm in a
smear of vaginal contents the following morning was considered as day zero of
pregnancy. The pregnant animals were divided into three groups.
The experimental group received a single 9-5 mg/kg i.p. injection of chlorambucil in 0-5 N sodium bicarbonate solution, pH 8-0 on the morning of day 14
and within 3 min after addition of drug to the buffer.
The second group, designated experimental controls, was injected with only
the buffer on day 14 and their food intake was restricted for the next 2 days
to mimic the weight loss of the drug-treated animals who had a 3-9 % body
weight loss the day after injection.
A third group consisted of untreated pregnant females allowed food and water
ad libitum. Their offspring were compared to experimental control fetuses with
every procedure and found not to differ. Therefore these two groups are
considered together as controls.
Phosphatases and abnormal skeletogenesis
205
Maternal animals were sacrificed on the morning of days 15-17 of gestation
by brief ether anaesthesia followed by cervical dislocation. For determination
of ion levels, maternal blood was drawn by cardiac puncture, allowed to clot,
and the serum removed. Amniotic fluid was collected by puncture of the fetal
membranes. Fetuses were removed from the uterine horn, the limbs were
removed into a cold Dounce homogenizer, and brought to a 10% weight/
volume homogenate with 0-25 M sucrose. Homogenization was accomplished
by five strokes each of a loose and a tight fitting pestle. Whole fetuses were
prepared in the same manner. All samples were centrifuged at 8000 g at 4°C
for 10 min and the supernatants removed for analysis. Aliquots were used for
protein and enzyme assays on the autopsy day. The remaining material was
stored frozen for later measurement of ions.
Phosphate was determined by colorimetric assay of acid-released phosphorus
(Gomori, 1942) while calcium and magnesium levels were determined on a
Perkin-Elmer atomic absorption spectrophotometer. Trichloroacetic acid
supernatants of the fetal homogenates was used to release these ions from the
extensive membranous fraction.
Acid and alkaline phosphatase activities were measured in aliquots of diluted
fetal limb homogenates incubated for 30 min at 37-5 °C in a total volume of 1 ml
containing a 0-05 M buffer (citric acid Na 2 HOP 4 for pH 4-0 to 6-4, tris/HCl for
pH 7-2-8-9, and sodium carbonate/sodium bicarbonate for pH 9-6-10-4) and
jD-nitrophenylphosphate as substrate. The reaction was stopped by NaOH and
color intensity measured spectrophotometrically (Bessey, Lowry & Brock, 1946).
Protein was determined by the method of Lowry, Rosebrough, Farr & Randall
(1951) and the phosphatase activity expressed as /miole of j3-nitrophenol
liberated per mg of protein.
The 1 ml reaction mixture for the assay of pyrophosphatase activity consisted
of 0-1 ml homogenate, 0-5 ml buffer, 0-5 HIM sodium pyrophosphate, and 7 0 mM
magnesium chloride. The system was at substrate saturation levels. After 15 min
incubation at 37 °C, the reaction was stopped by 0-5 ml 20% trichloroacetic
acid. The samples were centrifuged and the orthophosphate determined in the
protein-free supernant by a colorimetric assay of phosphorus based on Gormori's method (1942). Controls were run without addition of homogenate to
rule out release of phosphate from the substrate due to trichloroacetic acid in
the deproteinizing step. Phosphate from this source proved negligible. Other
samples were assayed without addition of substrate to verify that hydrolysis of
endogenous pyrophosphate was not detectable.
RESULTS
Acid and alkaline phosphatases
Limbs of day-15 experimental fetuses contained />-nitrophenylphosphatase
activity at a level similar to controls except at the most alkaline pH's where
14
EMB
53
206
E. S. BRUMMETT AND E. M. JOHNSON
1-6
1-4
1-2
I 10
%
n
0-8
0-6
0-4
0-2
7
10
S
11
pi I
Fig. 1. p-Nitrophenylphosphatase activity in limb homogenates of day-15 fetuses.
1-6
1-4
1-2
f,
o
™ 0-6
0-4
0-2
7
S
10
11
pi I
Fig. 2. jD-Nitrophenylphosphatase activity in limb homogenates of day-16 fetuses.
Figs. 1 and 2. Specific activity of pH-dependent phosphatases of limb homogenates
from control (O) and experimental ( • ) fetuses. Specific activity is expressed as
/tmole of phenol released per mg of protein in 30 min at 37 °C. Each mean in the
acid and alkaline peaks represents six or seven determinations. Vertical bars represent
standard deviations.
Phosphatases and abnormal skeletogenesis
207
1-6
1-4
1-:
io
0-8
0-6
04
0-2
7
8
9
10
II
pi I
Fig. 3. p-Nitrophenylphosphatase activity in limb homogenates of day-17 fetuses.
450
T
J
400
I
350
•
300
i
t
r 450
't
i 400
|
$
350
'i 300
r
500
i}
450
i •!
400
t
350
iT
300
t
I
f
t
I
80
8-5
L
70
7-5
pH
Fig. 4. Pyrophosphatase activity in limb homogenates from day-15 to day-17 fetuses.
Figs. 3 and 4. Specific activity of pH-dependent phosphatases of limb homogenates
from control (O) and experimental ( • ) fetuses. Specific activity is expressed as fig of
phosphorus released per mg of protein in 15 min incubation at 37 °C. Each mean
represents from two to four determinations.
14-2
208
E. S. BRUMMETT AND E. M. JOHNSON
Table 1. Ionic concentrations {/.tgj'ml)
10% Limb homogenate
Day 15
Control
Experimental
Day 16
Contro
Experimental
Day 17
Control
Experimental
10% Whole fetus
homogenate
Day 15
Control
Experimental
Day 16
Control
Experimental
Day 17
Control
Experimental
Maternal serum
Day 15
Control
Experimental
Day 16
Control
Experimental
Day 17
Control
Experimental
Amniotic fluid
Day 15
Control
Experimental
Day 16
Control
Experimental
Day 17
Control
Experimental
Magnesium
Calcium
Phosphate
Mean S.D.
Mean S.D.
Mean S.D.
40 (5) 29
40(3)16
11-7(3)0-8
11-9(3)1-9
9-9 (6) 0-2
10-5 (4) 0-2
56 (6) 35
58 (4) 16
12-9(6)0-6
17-7 (4) 3-5
9-9 (6) 0-2
9-7 (6) 0-2
74 (6) 33
98 (6) 60
17-2(5)0-8
17-9(5)3-9
10-3 (2) 0-4
10-4(1)
41 (2) 9
16-5(2)1-4
13-3(1)
100 (3) 0-4
9-6 (3) 0-4
42 (3) 19
34(3) 7
18-7(4)3-8
18-4(3)2-9
10-3 (3)0-8
9-9 (3) 0-4
59(3)18
51 (3) 12
23-8(3)2-1
21-1(3)3-7
23-7 (6) 3-1
23-6(5)4-2
100(5) 6
99(5) 8
69-4 (5) 8-7
62-6 (6) 8-4
24-3 (6) 2-5
21-3(5)3-5
102(6) 7
99(5) 8
75-9 (7) 7-9
74-9 (5) 70
21-3(5)30
230 (5) 30
102(5) 5
100(6) 4
72-4 (5) 60
72-1 (4) 7-5
27-5(8)1-4
27-4(6)1-2
92(8) 6
92(7) 5
42-4 (6) 7-2
45-3 (6) 6-9
240(7)1-2
26-6(5)2-1
92(7) 5
96(5) 5
36-5 (7) 6-2
42-8 (5) 7-9
23-2(6)1-1
23-8(5)1-0
90(6)10
91(4) 7
29-5 (5) 3-9
43-7 (5) 6-2
8-7 (5)* 0-4
90(3)1-3
* Number of determinations (litters).
Phosphatases and abnormal skeletogenesis
209
Table 2. Protein concentration of amniotic fluid (mg/ml)
Day 15
Mean*
s. D.
Day 16
Mean
S.D.
Day 17
Mean
S.D.
Control
1-79
017
1-94
013
186
017
Experimental
1 -91
0-27
2-58
017
2-50
0-24
* Each mean is an average of four determinations, samples from one litter having been
pooled for a determination.
activity was significantly (P < 0-01) lower (Fig. 1). Both groups had a similar
peak of activity between pH 5-5 and 5-8. On day 16, the limbs of both the
control and drug-treated fetuses retained the acid phosphatase peak seen earlier
and both now had more activity in the alkaline range (Fig. 2). Activities were
significantly different (P < 0-01) only at pH 9-8. By day 17, the acid phosphatase
peak had increased slightly in both groups (Fig. 3). In the alkaline range, the
control tissue's enzyme activity had increased about 55 % over the previous
days' values, while in experimental tissue the increase was approximately 40%.
The specific activity of the enzyme from the two groups differed significantly
over most of the alkaline range (between pH 8-5 and 10-4).
Pyrophosphatase
Incubation media with pH's of 5-3-10-0 were used for the assay of pyrophosphatase but activity fell to barely detectable levels at either pH extreme.
The pH curves for the 3 days were similar for both normal and abnormal limbs
but (Fig. 4) consistently lower mean levels of pyrophosphatase activity in the
limbs of abnormally developing fetuses were seen.
Ion levels
Phosphate and calcium levels increased slightly in both groups with increasing gestational age; however, there was no significant difference between control
and experimental limbs in their phosphate, calcium or magnesium concentrations
over the 3 days under study (Table 1). This same pattern was seen in homogenates of entire fetuses as well. Maternal serum phosphate, calcium and
magnesium concentrations were similar in drug-treated and control dams and
no fluctuations were seen over the 3 days of pregnancy examined (Table 1).
Calcium and magnesium concentrations remained unchanged and at comparable levels in the amniotic fluid from both groups of fetuses on all 3 days but
ammiotic fluid collected from experimental fetuses on day 16 contained a slightly
higher level of phosphate than normal and the level was significantly higher by
day 17 (Table 1). The protein content of amniotic fluid from experimental fetuses
was significantly higher than controls (P < 0-1) on days 16 and 17 (Table 2).
210
E. S. BRUMMETT AND E. M. JOHNSON
Amniotic fluid volume was measured and found to be similar in both treatment
groups.
DISCUSSION
A previous histological study of limbs from chlorambucil-treated fetuses
(Brummett & Johnson, 1979) has shown that on day 15, the day after drug
exposure, the long bone primordia exist as cartilage models. By day 17, the
normal cartilage models have extensive calcified areas, some bone marrow
formation and beginning periosteal mineralization. Drug-treated limbs at this
age have much less calcification of the hypertrophic zone matrix, chondrocytes
are of abnormal size and arrangement, and bone marrow formation is at only
the initial stage in most limb bones.
The chlorambucil-treated rat fetuses provide a convenient system for the study
of biochemical events related to abnormal skeletogenesis as they have consistent
skeletal malformations accompanied by minimal fetal death (Brummett &
Johnson, 1979). Tissue that is not grossly recognizable as abnormal can be
pooled at day 15 without dilution by excessive normal tissue.
Assay of fetal limbs for enzyme activity demonstrated that the acid phosphatase activity remained unchanged and at a comparable level for both the
normal and drug-treated limbs over the 3-day period. Acid phosphatase is
present to a marked extent in osteoclasts but apparently the enzyme is not
involved in the bone malformations of the experimental fetuses.
The alkaline phosphatase activity was low on day 15 in both normal and
abnormal tissue since very few areas of the limbs are undergoing calcification.
The failure of experimental limbs to increase in alkaline phosphatase activity
on day 16 and day 17 to the same extent as normal limbs, could reflect a
decreased osteoblast population since an ultrastructure study has localized
significant amounts of alkaline phosphatase in the cell membranes of osteoblasts
and chondroblasts (Gothlin & Ericsson, 1971). Because enzyme activity is in
terms of mg protein it would necessarily imply replacement of normal osteoblasts by undifferentiated or abnormal cells having less enzyme and not just a
decrease in total cell number. Jaffe & Johnson (1973) also found depressed levels
of alkaline phosphatase activity in abnormally developing limbs from fetal rats
of folic acid-deficient dams and (Krotoski & Elmer, 1973) reported reduced
amounts in limbs of brachypod fetal mice, whose mutant gene causes abnormal
formation of the cartilage in the zone of hypertrophy.
Ionic concentrations of limb and fetal homogenates did not reflect the
decreased calcification in abnormal fetuses. Phosphate and calcium tended to
increase in both groups. Possibly, in treated fetuses, other tissue conditions
weren't appropriate for mineralization or the ions were not in an appropriate
form or site.
The amniotic fluid of experimental fetuses during skeletal differentiation
differed from normal on day 17 with an increased level of phosphate and
Phosphatases and abnormal skeletogenesis
211
protein. Since phosphate is mainly an intracellular ion, elevated concentrations
of both the substances could be due to cell damage and loss from the fetus or
its membranes.
Although no alterations in the normal concentrations of phosphate, calcium
and magnesium were detected in the abnormal fetuses, inadequate levels of
phosphate in matrix vesicles, the probable site of calcification, cannot be ruled
out. The large increase in alkaline phosphatase activity in normal fetal limbs
from day 15 to 17 is definitely associated with the onset of mineralization. The
much smaller percentage of increase in the drug-treated limbs provides a useful
model for studies of skeletal formation and abnormal fetal development.
REFERENCES
An, S. Y., SAJERA, S. W. & ANDERSON, H. C. (1970). Isolation and characterization of
calcifying matrix vesicles from epiphyseal cartilage. Proc. natn. Acad. Sci., U.S.A. 67,
1513-1520.
ANDERSON, H. C. (1969). Vesicles associated with calcification in the matrix of epiphyseal
cartilage. /. Cell. Biol. 41, 59-72.
ARSENIS, C , RUDOLPH, J. & HACKETT, M. H. (1975). Resolution, purification and characterization of the orthophosphate releasing activities from fracture callus calcifying cartilage.
Biochim. biophys. Acta. 391, 301-315.
ARSENIS, C , HACKETT, M. H. & HUANG, S. M. (1976). Resolution, specificity and transphosphorylase activity of calcifying cartilage alkaline phosphatases. Calcif. Tiss. Res. 20,
159-171.
BESSEY, O. A., LOWRY, O. H. & BROCK, M. J. (1946). A method for the rapid determination of
alkaline phosphatase with five cubic millimeters of serum. /. biol. Chem. 164, 321-329.
BONUCCI, E. (1967). Fine structure of early cartilage calcification. /. Ultrastruct. Res. 20,
33-50.
BRUMMETT, E. S. & JOHNSON, E. M. (1979). Morphological alterations in the developing fetal
rat limb due to maternal injection of chlorambucil. Teratology, (In the Press.)
FELIX, R. & FLEISCH, H. (1976). Pyrophosphatase and ATPase of isolated cartilage matrix
vesicles. Calcif. Tiss. Res. 22, 1-7.
FLEISCH, H. & RUSSELL, R. G. G. (1970). Pyrophosphate and polyphosphate. Int. Enc.
Pharmacol. Ther. 1, 61-100.
FRANCIS, M. D. (1969). The inhibition of calcium hydroxyapatite crystal growth by polyphosphates. Calcif. Tiss. Res. 3, 151-162.
GOMORI, G. (1942). A modification of the colorimetric phosphorus determination for use with
the photoelectric colorimeter. /. Lab. Clin. Med. 27, 955-960.
GOMORI, G. (1943). Calcification and phosphatase. Amer. J. Path. 19, 197-207.
GOTHLIN, G. & ERICSSON, J. L. E. (1971). Fine structural localization of alkaline phosphomonoesterases in the fracture callus of the rat. Israel J. med. Sci. 7, 488-494.
JAFFE, N. R. & JOHNSON, E. M. (1973). Alterations in the ontogeny and specific activity of
phosphomonoesterases associated with abnormal chondrogenesis and osteogenesis in the
limbs of fetuses from folic acid-deficient pregnant rats. Teratology 8, 33-50.
KROTOSKI, D. M. & ELMER, W. A. (1973). Alkaline phosphatase activity in fetal hind limbs of
the mouse mutation brachypodism. Teratology 7, 99-106.
LOWRY, O., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement
with the folin phenol reagent. /. biol. Chem. 193, 265-275.
MAJESKA, R. & WUTHIER, R. E. (1975). Studies on matrix vesicles isolated from chick
epiphyseal cartilage. Association of pyrophosphatase and ATPase activities with alkaline
phosphatase. Biochim. biophys. Acta 392, 51-60.
SALOMON, C D . (1974). A fine structural study on the extracellular activity of alkaline
phosphatase and its role in calcification. Calcif. Tiss. Res. 15, 201-212.
(Received 4 July 1978, revised 30 March 1979)