STUDIES ON THE CHEMICAL COMPOSITION OF

BONE ASH
BY SERGIUS MORGULIS
WITH THE TECHNICAL ASSISTANCE OF E. JANECEK
(From the Marine Biological Laboratory, Woods Hole, and the Department of
Biochemistry, College of Medicine, University
of Nebraska, Omaha)

(Received for publication, June 3, 1931)

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An understanding of the composition of bone is essential for the
proper interpretation of the process of its formation in the living
organism. It is obvious that definite physicochemical conditions
are necessary to promote bone formation from components dis-
solved in the circulating fluid of the body. But it is still a moot
question whether the physicochemical condition is the only factor
or merely one of the essential factors determining the process.
Viewed from the strictly physicochemical standpoint, the problem
may be said to be in total confusion. Is bone formation simply a
precipitation of insoluble salts and, if so, is this associated with a
state of supersaturation of the serum with Caa(PO&, as is believed
by some (3, 4), or with a state of undersaturation with CaHPO+
as is assumed by others (16, 17)? “All these conclusions,” as is well
pointed out by Floseberry, Hastings, and Morse, “may be inde-
pendently correct and yet have no relation to the situation exist-
ing between serum and bone.” Is the basic salt complex of bone a
compound of the nature of Ca[ { Cas(PO& 1&Os (Gassmann (2))
or of Ca[ ( Cas(PO&) s](OH)z (Klement (8, 9)) and, if so, does it
possess a definite crystalline structure? On neither of these funda-
mental questions has a final answer yet been definitely obtained.
The recent contribution of Robison and collaborators (12, 13)
again brings out the significance of an enzyme mechanism for
creating a condition of supersaturation in the matrix fluid neces-
sary for calcification, and the possibility that phosphate esters
may play an important role in the process of bone formation.
455
456 Chemical Composition of Bone Ash

The contribution which this paper attempts to make to this

controversial subject deals with a study of the balance of basic and
acid equivalents in the bone ash. It is based upon an analysis
of the mineral composition of the vertebrae in a variety of animal
forms from fishes to mammals.
Analytical Results
The vertebrae, freed from soft tissue as nearly as possible, and
defatted with boiling alcohol, were ashed by the process proposed
by Gabriel (1) but with certain important modifications. The
method consists in dissolving the entire organic matrix of bone by

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heating in a 3 per cent KOH solution in glycerol. We found it,
however, essential to use a reagent previously freed from water by
boiling and we carry out the ashing by heating the bone at about
250”. The bone thus leached out with alkaline glycerol reagent
retains its morphological form perfectly, only the organic matter
being destroyed and leaving a compact white mass of bone salts.
Our procedure of washing the ashed bone is also an important
departure from Gabriel%. The leached bone is boiled briskly
with distilled water once or twice, depending on how much of the
reagent adheres to the surface; it is then extracted several times
in boiling 95 per cent alcohol, to remove as nearly as possible all
the glycerol reagent, and is dried to constant weight at 110”.
The bone in this condition can be easily reduced to a fine powder;
indeed it crumbles when pressed between the fingers. The bone
powder which may be snowy white or only slightly tinged, dis-
solves in hydrochloric acid without leaving any insoluble residue.
The analyses were made on bone ash powders thus prepared. A
weighed amount of powder dried to constant weight is dissolved
in a measured quantity of standard HCl, calcium, magnesium,
potassium, and phosphate being determined in aliquot portions
of this solution by the usual procedures. Calcium was precipi-
tated as ammonium oxalate, which was dissolved in sulfuric acid
and titrated with 0.005 N KMn04. The phosphates were de-
termined by the Fiske-Subbarow calorimetric method. However,
all the phosphate determinations were subsequently repeated by
two other procedures; gravimetrically, by precipitating the phos-
phate as strychnine phosphomolybdate and also, volumetrically,
by Fiske’s method. Except for one or two instances the results
 S. Morgulis 457

by all three methods checked perfectly and where a discrepancy

occurred between the calorimetric determination and the others,
the result of the gravimetric analysis was accepted. The analyses
of the gravimetric method were accurate within f 0.2 per cent.
The magnesium was precipitated as MgNH4POr and the phosphate
determined calorimetrically. Owing to the very small amounts of
potassium present in the bone material the direct determination
was not possible. We determined, therefore, the potassium by
Kramer’s method in a solution of known potassium content and
in a similar sample to which an aliquot of the bone ash solution
was added. The potassium of the bone ash was calculated from

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the difference between the two analyses. The carbonate was
determined in weighed amounts of bone ash powder in a modified
Van Slyke COz apparatus according to Kramer’s procedure (15).
In addition to the above analyses, an aliquot portion of the
bone ash solutions was titrated with standard sodium hydroxide,
methyl orange being used as an indicator, or, after boiling
off the CO*, by titrating electrometrically to the pH of a KH2P0,,
solution, The titrations by both procedures were the same and
we, therefore, limited ourselves to the indicator titration only.
The first question which naturally presents itself is whether the
treatment with the glycerol reagent does not alter the composition
of the bone salts in some essential respect. We tested this point
in two ways and feel satisfied that the procedure is unassailable.
First, we analyzed a sample of tricalcium phosphate dissolved in
hydrochloric acid, determining the Ca and P by the usual methods
and from the results a Ca: P ratio of 1.92 was calculated for this
material. Another sample of the same material was then sub-
jected to the treatment with the alkaline glycerol, exactly as was
done in the case of the bone material, the Ca and P analyses giving
a Ca:P ratio of 1.92, just as for the untreated salt. Secondly, we
analyzed untreated powdered bone and a sample of the same bone
which had been leached out by the glycerol reagent. The results
of the analyses of the two samples are recorded in Table I from
which it is obvious that, correcting for the organic content of the
untreated bone, boiling with the alkaline glycerol, and subsequent
extraction with boiling alcohol does not affect the composition of
the mineral components of the bones. The values for Ca and P
are perhaps somewhat higher as determined on the bone ash
458 Chemical Composition of Bone Ash

material than on the original unashed whole bone. However,

the untreated bone powder was extracted with trichloroacetic acid
according to Kramer’s method of bone analysis (15), and this may
account for the small differences. It is possible, of course, that
the calcium was not completely extracted and, as far as the phos-
phate determination is concerned, the trichloroacetic acid extract
of bone is decidedly less favorable than the bone ash solution for
calorimetric analysis, the color in the former instance being
greenish blue and difficult to match, whereas in the latter instance
a beautiful pure blue color develops.
The results of our analytical study are presented in Tables II

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and III, in which the elementary composition of the ash and the
balance of acid and basic equivalents, respectively, are given.
In calculating the bone salt composition from the elementary

TABLE I
-
Ash Whole bone (60.7 per Ratio
cent organic matter)
-
per cent per cent
Ca 32.96 15.89 1.02
P 15.75 7.68 1.01
cot 2.32 1.17 0.98
ChCOJ 5.28 2.66 0.98
Cas(PO& 79.55 38.65 1.01
-

analysis certain assumptions have been made. First, that the

magnesium in the bone ash is present entirely in the form of
Mgs(PO&; secondly, that the predominant part of the potassium
is in the form of KOH. Of course, some of the magnesium may
be and probably is found as carbonate, but it does not affect the
calculation materially even if the opposite assumption were made;
namely, that all the magnesium in the bone ash is in the form
of MgC03. As regards the potassium, although it is found in
bone tissue, it seemsvery doubtful if it is really a component of the
bone salts. More likely it is there accidentally, as an admixture
from the body fluids. In bone ash preparations made by leaching
the organic matrix with the alkaline glycerol reagent the KOH is
undoubtedly an impurity. If we take Weiser’s (19) analysis of
bone ash obtained by incineration, we may assume that bone
 S. Morgulis
might contain approximately 0.2 per cent of potassium as a con-
stant component and that the considerably higher values obtained
in our analyses must be attributed to small amounts of KOH
from the reagent which were not completely washed out. Should
we even allow that about 0.2 per cent is present originally in the
bone mineral matter in the form of K&OS this correction again
would have no material effect upon the calculated composition.
Furthermore, as far as the acid-base balance of the bone ash is
concerned, this is not at all affected by either assumption.
Examining the data of the analyses recorded in Table II, giving
the composition of vertebrae obtained from a variety of animals

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beginning with the cartilaginous fish (dogfish) and extending
through the bony fishes, amphibians, reptiles, birds, and mammals,
one is impressed by the remarkable similarity of the bone ash
composition. The most striking difference which manifests
itself in this series of comparative analyses is the relatively ow
CaCOs content of the vertebrae from marine fishes, with a
Car(PO&:CaCOa ratio which in round numbers is 12: 1, whereas
in all the other animals studied, owing to the considerably higher
carbonate content, this ratio is much lower and is on the average
6.6:1. The low CaCOs content of the skeletal structure of ma-
rine fishes is probably due to the extremely low bicarbonate
reserve of their body &ids. The residual calcium values (Ca
other than CaCO,) do not bear the relation to those of the residual
P values (total P minus P in the form of Mgs(PO&) as would be
expected if the res dual substance were nothing but Ca8(P04),.
In the latter csse, the theoretical Ca:P ratio would be 1.935
whereas the ratios found invariably in the analyses of the salts
of the vertebrae range from 1.99 to 2.12. In other words, a certain
amount of calcium found in the analyses cannot be accounted for
either as carbonate or phosphate. The calculated excess of Ca
is recorded in the last column of Table II.
Turning now to Table III, we observe that the total basic
equivalents of the ash exceed the total acid equivalents, 88 shown
in Column 8. In general there are about 19 basic to 18 acid
equivalents. Although neither Cl nor F was determined, both
these radicles are found in such minute amounts in bone ash that
their presence may be neglected without affecting the equivalent
balance. Similarly the data obtained from titrating the bone
460 Chemical Composition of Bone Ash

ash so]utions corroborate the presence of such an excess of a basic

component in the bone ash. The data recorded in Columns 9,
10, and 11 of Table III require a word of explanation. On dis-
TABLE II
Elementary Composition of Bone Ash Expressed in PeT Cent

Composition of bone ash Calaulated composition

e!
Material .s ” .a d 0

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2- 8I 2 2s 23 i
5 8w & 8 g 6 v2 59
60 2 r4
-----------
Dogfish 37.000.580.4416.713.232.09 7.3480.00 10.9 2.123.06
Gooeefieh 36.900.550.5616.983.191.99 7.2782.50 11.4 2.052.08
Mackerel 36.740.760.2017.422.792.74 6.3583.85 13.2 2.041.90
Squeteague 35.680.621.3116.973.111.96 7.0482.20 11.6 2.001.03

Frog (common) 36.090.720.4416.454.672.5910.6279.15 7.5 2.011.20

Bullfrog 36.620.610.3616.154.112.20 9.3578.10 8.1 2.102.63
Amphiuma 36.860.510.7615.965.341.8412.1377.60 6.4 2.062.01
Siren 36.960.571.0516.005.732.0613.0277.55 6.0 2.051.75
Turtle 35.680.620.9814.885.632.2412.8371.75 5.6 2.132.75
Hen 37.240.51 16.405.501.8312.5079.80 6.4 2.021.46
Rabbit 36.250.530.9215.995.711.9112.9977.65 6.0 1.991.00
cow 36.050.740.8516.434.582.6610.4078.95 7.6 2.021.30
Dog 35.660.461.8715.565.621.6412.7875.80 5.9 2.031.15
Guinea pig 35.540.75 15.785.032.7111.4475.65 6.6 2.041.66

Thyroid calculi 34.40 0.71 15.364.772.5510.8573.75 6.9 2.031.50

34.600.63 15.804.512.2710.2276.30 7.5 2.000.91

Mixtures of tri- 37.70 15.855.83 13.2579.25 6.0 2.03 1.70

calcium phos- 36.44 15.644.83 11.0078.20 7.2 2.05 1.89
phate (Eimer 37.18 16.80 3.04 6.9284.00 12.1 2.001.86
and Amend)
and calcium
carbon&e

solving the bone ash in a measured volume of 0.1 N HCl the fol-
lowing reactions take place.
CaCOJ + Car(PO& + 8 HCl = 4 CaCL + 2 HsPOC + H&O8
 TABLE III
Balance of Acid and Basic Radicles of Bone Ash
Equivalenta per loo0 gm. ash Used litem of N HCI per 1000 gm. ash
- - - -
Mabxial ( 1Di(slT*c
Ca Mg E PO4 co2 ‘otal barn e’l Ma1 acb I’otd ash 1
I I
(1) (2) (3) (4) (5) (‘3 (7) (8) (9) (W
__ . - __
Dogfish 18.50 0.48 0.11 16.16 1.47 19.09 17.63 1.46 13.29 12.36 0.93 0.53
Goose&h 18.45 0.46 0.14 16.15 1.45 19.05 17.60 1.45 12.87 12.36 0.51 0.94
Mackerel 18.37 0.63 0.05 16.83 1.27 19.05 18.10 0.95 12.90 12.54 0.36 0.59
Squeteague 17.84 0.52 0.34 16.40 1.42 18.70 17.82 0.88 13.13 12.70 0.43 0.45

Frog 18.05 0.60 0.11 15.89 2.12 18.76 18.01 0.75 13.49 12.83 0.66 0.09 r/l
Bullfrog 18.31 0.51 0.09 15.88 1.87 18.91 17.75 1.16 12.98 12.54 0.44 0.72
Amphiuma 18.43 0.42 0.19 15.57 2.45 19.04 18.02 1.02 13.27 13.02 0.25 0.77 k
Siren 18.48 0.47 0.27 15.47 2.61 19.22 18.08 1.14 13.44 13.20 0.24 0.90 g
Turtle 17.84 0.52 0.25 14.35 2.55 18.61 16.90 1.71 13.35 12.36 0.99 0.72 $
Hen 18.62 0.42 0.20 16.33 2.50 19.24 18.83 0.41 13.73 13.59 0.14 0.27 r
Rabbit 18.12 0.44 0.23 15.28 2.59 18.79 17.87 0.92 13.65 13.01 0.64 0.28 a
cow 18.03 0.62 0.21 15.88 2.08 18.86 17.96 0.90 13.45 12.88 0.57 0.33
Dog 17.83 0.36 0.48 14.95 2.53 18.69 17.50 1.19 13.09 13.00 0.09 1.10
Guinea pig 17.77 0.63 0.20 15.78 2.29 18.60 18.07 0.53 13.09 13.01 0.08 0.45

Thyroid calculi 17.20 0.59 0.20 14.85 2.17 17.99 17.02 0.97 13.07 12.87 0.80 0.17
17.30 0.63 0.20 15.56 2.05 18.13 17.61 0.52 13.06 12.63 0.43 0.09

Mixtures of trical- 18.85 15.36 2.64 18.85 18.00 0.85 13.78 12.88 0.90 0.05
cium phosphate 18.22 15.11 2.20 18.22 17.33 0.89 12.49 12.28 0.21 0.68
(Eimer and 18.59 16.70 1.38 18.59 18.08 0.51 13.30 12.52 0.78 0.27
Amend) and cal-
cium carbonate
_- - - -

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462 Chemical Composition of Bone Ash

In addition to these reaction products the mixture, of course, will

contain the unused excess of 0.1 N HCl. In titrating this with
0.1 N NaOH to methyl orange 1 equivalent of the H3P04 is de-
termined together with t-he excess HCl. In Column 9 the titra-
tion results are recorded calculated in terms of N HCl reacting
with 1000 gm. of the bone ash. Knowing from the a.nalyses the
total carbonate and phosphate content of the dissolved material,
one can easily calculate t,he required quant&y of HCl. Allowance
is also made for the extra base in the form of I<OH. The calcu-
lation can be best illustrated by an example.

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250 mg. bone ash dissolved in,. 50.00 cc. 0.1 N HC1
0.1 N NaOH required for titrating back to
methyl orange........................... 15.70 “
Used up by 250 mg. bone ash.. 34.30 1. 0.1 N HCl
“ IL C‘ 1000 gm. “ ” . 13.72 ” N HCl
Acid used up to dissolve carbonates.. 2.50 ”
11.22 ” N HCl
Acid used up to dissolve phosphates (f equiv-
alent).. _. 10.88 ”
0.34 “ N HCl
Acid used up bo neutralize KOH.. _, 0.20 ”
Excess acid used up. _. 0.14 “ N HCI

In this particular instance the analysis of the bone ash showed

an excess of basic over acid radicles of 0.41 equivalent. In
Column 10 of Table III t,he total correct,ion for carbonates, phos-
phates, and KOH is recorded as a single value to save space.
It will be noted that the titrations indicate consistently the
presence of a basic component in the bone ash, the acid used up
in the titrations being invariably greater than demanded by
the reaction. We have aheady seen that the analyses of the
ash for Ca a.nd P also show an excess of the former, if the Ca and
P were components of a substa,nce like Ca3(PO& To be sure,
t,he excess basicity of the ash, as determined by titration, and
the excess Ca as determined from the a.nalysis, are not exactly
equivalent to each ot,her. Nevertheless, when one bears in mind
that the inherent errors instead of compensating may be and fre-
quently are cumula.tive, the coincidence of the results of direct
analysis and of calculation from titration is most significant.
Similar results were also obtained from the study of the calcified
 S. Morgulis 463

masses from the thyroid gland as reported in Tables II and III.

These facts point unavoidably to the conclusion that biological
calcareous structures contain a basic calcium compound in addi-
tion to the other components, Ca3(PO& and CaC03.

DISCUSSION

When the various view-points regarding the nature of the bone

salts are surveyed, certain principal trends can be recognized.
The oldest conception, first proposed by Hoppe (5) and later
elaborated by Werner (20), assumes the existence in bone of a
complex salt consisting of calcium carbonate and phosphate,

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Ca[( Ca (POd213lCO3, which places it in the apatite series of
minerals. de Jong (7), Taylor and Sheard (18), and recently also
Roseberry, Hastings, and Morse (14) by means of x-ray spectrum
analysis of bone material brought forth evidence in favor of the
above hypothesis of the apatite crystalline structure of bone salts,
as was originally postulated by Hoppe. Taylor and Sheard
even consider the crystal structure of the bone to be closely rela-
ted to that of the apatite mineral podolite. Another view-point
postulates that the bone salts may be represented as a combina-
tion of secondary CaHP04 and CaO, or a basic secondary phos-
phate (17). However, there is no evidence of the existence of
CaHP04 in the bone ash. Taylor and Sheard and Roseberry,
Hastings, and Morse pointed out that their studies fail to support
such an assumption. Apart from such lack of support from
physical studies on bone, the chemical composition of the bone
ash cannot be brought into agreement with the CaHP04 hypothe-
sis. Furthermore, if CaHP04 were present in bone it would upon
incineration be converted to pyrophosphate and could not be
determined until it had been changed back to orthophosphate by
acid hydro!ysis. This, however, we did not find to be the case
with our bone ash material. On the contrary, when (NH&HP04
is precipitated slowly and with constant stirring with CaCL,
while maintaining a practically constant pH of 7.5 by the addition
of sodium hydroxide, we obtained a substance which was proved
to consist of Ca3(PO& and CaHP04 and which by analysis was
found to contain about 14 parts by weight of Ca3(PO& and 1
part of CaHPO+ giving thus a molar ratio of 6.0: 1.0.
Does the bone ash represent a complex calcium phosphate-
464 Chemical Composition of Bone Ash
carbonate salt? In spite of various claims in favor of its existence
based upon investigations by physical methods (7, 14, lS), the
results of chemical analysis do not support such a view. In the
first place, as we have found in the series of comparative analyses,
the Ca3(P0J2:CaC03 ratio is radically different in marine fishes
than in all other animals examined, the low calcium carbonate
content of their bone ash bearing a very suggestive relationship
to the extremely low bicarbonate reserve of the blood in those
fishes. If bone ash were a definite crystalline compound of the
carbonate and phosphate of calcium, its formation might be has-
tened or delayed by variations in concentration of the essential

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components dissolved in the blood serum, but would this affect
the composition of the crystal? Furthermore, the phosphate:
carbonate ratio does not correspond to the theoretically expected
ratio of 9.3 and is variable, being apparently influenced by different
physiological conditions (it is markedly lowered by rickets (6) and
is altered by the state of nutrition, by age, and by phosphorus
deficiency in the diet (10,ll)).
Our analytical studies bring out the significant fact that bone
ash contains more calcium than can be accounted for on the basis
of carbonate and phosphate and, furthermore, that this excess of
calcium must be in the nature of a basic compound which uses up
acid in the titration. Our method of preparation of the bone ash
precludes the possibility that this excess might be derived from Cn
collagenate of the bone matrix. In this respect the bone ash com-
position is similar to that of a tricalcium phosphate (Eimer and
Amend, Tested Purity grade) which on analysis also showed an
excess of 1.64 equivalents of Ca and which by our titration pro-
cedure was found to be a basic Ca. This purchased tricalcium
phosphate was, therefore, quite different from the material which
we ourselves prepared in that it contained Ca(OH)* instead of
CaHPOl although in the same molar ratio.
Turning once more to the data reported in Table II and guided
by the evidence of the presence of a basic calcium compound in
the material, we may now attempt to determine the composition
of the bone ash on the basis of this chemical information. For
this purpose the average value for separate groups rather than for
each separate sample will be considered. From the results of the
analyses of the bone ash of marine fishes the average composition
 S. Morgulis 465

corresponds to 82.15 per cent C&(PO&, 7.00 per cent CaC03, and
3.74 per cent Ca(OH)t. In terms of mols these values are 82.15:
310, 7.00: 100, and 3.74: 74, thus giving a molar ratio of 6.00:
1.57: 1.13 which is most nearly satisfied by the composition
Ca[(Ca3(P04)2)6l(OH)z. Similarly, from all the other bone ash
analyses an average composition is obtained of 77.20 per cent
Ca3(PO&, 11.81 per cent CaC03, and 3.13 per cent Ca(OH)2. This
corresponds to a molar ratio of 6.00 : 2.82: 1.02, which satisfies the
above formula even more perfectly. For the artificial mixtures
of CaC03 and Cas(PO& (Eimer and Amend) the composition
corresponds to a molar ratio of 6.00:2.86: 1.06, for the first two,

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and of 6.00: 1.53: 1.04 for the third mixture, for the Ca,(PO&,
CaC03, and Ca(OH)2 respectively. In other words, the Eimer
and Amend tricalcium phosphate product, like the bone ash
material, seems to be correctly described by the formula
Ca[{Car(P04)2}6](OH)z. It may be recalled that the tricalcium
phosphate which we ourselves prepared contained CaHP04, the
molar ratios being practically 6.00 : 1 .OO, so that it likewise might
be described as the following compound, Ca [ ( Ca3(PO&) 6]HPO+
in which CaHP04 takes the place of the Ca(OH)2.
It is not easy to apply this mode of calculation to bone ash
analyses reported by other authors owing to the fact that essential
data are generally lacking as, for instance, the COz content, where
the analyses have been made on incinerated bone. We have
taken, however, one of Shear and Kramer’s analyses on whole
bone material ((15) p. 114) and, introducing a correction for
magnesium, we find that the composition of that particular sam-
ple of bone was 51.00 per cent Ca3(P0&, 9.21 per cent CaC03,
and 2.09 per cent Ca(OH)2. The latter was calculated from the
calcium not accounted for as carbonate and phosphate. This
relative composition corresponds to a molar ratio of 6.00:3.30:
1.00, thus corroborating in a general way what we have found in
our analyses of bone ash.

SUMMARY AND CONCLUSION

A comparative investigation of the vertebrae has shown that

the principal difference between the chemical composition of the
bone ash in marine fishes and in a variety of higher vertebrates is
in the proportion of CaC03, which is only about one-half as large
as in the latter.
466 Chemical Composition of Bone Ash
Evidence is adduced to show that the bone ash does not contain
CaHP04. However, the bone salts do not consist merely of
CaC03 and Ca3(PO& but very probably also of Ca(OH)2. Since
analyses do not reveal any definite and simple molar relationship
between the CaC03 and Ca&PO& the hypothesis that a crystal-
line complex salt Ca [ { Ca3(PO&) 3]C03 is the basis of bone structure
seems improbable. On the contrary, a definite and simple molar
relation between the Ca3(P0& and Ca(OH)2 of 6.00: 1.00 does
point strongly to the possibility that the principal component of
the bone ashis acomplexsalt of the nature Ca[ ( Ca3(P04)216](OH)2.

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 STUDIES ON THE CHEMICAL
COMPOSITION OF BONE ASH
Sergius Morgulis and With the technical
assistance of E. Janecek
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