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THE COMPARATIVE UTILIZATION
OF ORTHO AND PYRO PHOSPHATE
IN BONE DEVELOPMENT OF RATS

Thai: for the Degree of M. S.
MICHIGAN STATE COLLEGE

Wilham H. Lynch
I942

THE COMPARATIVE UTILIZATION OF ORTHO
AND PYRO PHOSPHATE IN BONE
DEVELOPMENT OF RATS

by .'

William.H§ Lynch

\

A.THESIS

Submitted to the graduate school of Michigan
State College of Agriculture and Applied
Science in partial fulfilment of the
requirements for the degree of
MASTER OF SCIENCE

Department of Chemistry

19h2

C H 141 r-na'rwv DEPT.

ACKNOWLEDGEMENT

The writer wishes to acknouledge his deep
appreciation and gratitude to Dr. C. A. Hoppert
for his suggestions and guidance in the planning

and deve10pment of this project.

TABLE OF CONTENTS

Historical Review -------------------------- 1
Experimental Part I - ---------------- -- ------ a
Comparative utilization of pyro and
ortho phosphate in bone deveIOpment
Data --.---------’---------~---°----------- 6
Discussion ------------------------ , --------- 8
Experimental Part II ------------------ ---~- 10
Specificity of Youngberg procedure
and site of conversion of pyro to

ortho phosphate

 

Summary
81b110graphy ------------------------------ 1h

Detailed Data --------- ,--- ................. 15

HISTORICAL

Ortho phosphoric acid and the various salts of this
acid are known to supply phosphorus for the various needs
of the body, the major ones being for the normal develop-
ment of bones and teeth. Thus for example ortho phosphates
are selected to supply the major portion of the phosphorus
of synthetic experimental diets. In addition to these
sources foodstuffs, of course, contain much of their
phosphorus in the form of phOSphorylated organic compounds
which on hydrolysis yield ortho phosphate.

Pyro phOSphates have been used for over twenty years
as an ingredient of leavening agents and interest in
their uholesomeness has recently been aroused in
connection with the revisions in federal food and drug
regulations. A persual of the literature reveals that
relatively little work has been done on the study of the
fate of pyrophosphates in the animal body. Gamgee,
Priestly, and Larmouth1 assumed that perphosphate passes
unchanged through the body and by intravenous injections
of the normal salt into rabbits and frogs demonstrated
that the pryOphosphate has a definite toxic effect,
causing a fall in blood pressure and a variation in
pulse rate, with continuous injections leading to death.‘
However, they do note the following: "We have never

succeeded in producing symptoms of poisoning by sodium

pyro phosphate when we have introduced the substance into
the stomach. In dogs the drug has produced vomiting if
fed in large amounts but no further ill effect; rabbits
have remained unaffected by it. So striking was the absence
of poisonous effects that it appeared to us possible

that some of the alimentary ferments might cause sodium
pyro phosphate to combine with water and convert it to
the inert ortho phosphate. Direct experiments with
salivary, gastric, and pancreatic ferments digested with
pyro phosphate at the temperature of the body, proved
that our surmise was not correct. At present we are
inclined to think that the fact of animals not being
poisoned when sodium pyro phosphate is introduced into
the alimentary tract is due to the rapid elimination of
the drug.“ In comparison Gamelz, in 1911, noted that the
pyro phOSphate goes over to the ortho phosphate in an

organism. Gardner andSymes3 working with cats

demonstrated that a small intravenous injection of the
tetra sodium pyro phosphate causes a fall in arterial
pressure and a slightly larger dose proves fatal. It

was at one time thought that the injurious effect of

cotton seed oil was due to the presence of pyro phosphate.“
In this work the supposed harmfulness of the pyro phosphate
was demonstrated by injecting an extract of the cotton

seed meal into the blood stream of a rabbit. Later

investigations showed that the oral administration of

this same extract had no effect on the animals.

Starkensteins believed that the conversion of
pyro phosphate to ortho phosphate takes place in the
stomach, due to the presence of hydrochloric acid.

Data obtained from the Victor Chemical Works indicates
that the hydrolysis of pyro phosphate in the presence

of .l N hydrochloric acid proceeds at a relatively slow
rate, less that 70% being converted to ortho phosphate
in a period of eight days. Inasmuch as food rarely
remains in the stomach for more than six to eight hours,
it is obvious that relatively little conversion would
take place in this region of the digestive tract.

Lohman6 has determined that the pyro phosphate is
transformed to the ortho phosphate in an organism, although
the site of such change was not suggested.

0n the basis of experimental data of Behrems and
Seelkoph7 who fed sodium pyro phOSphate to male mice
it was demonstrated that ingestion of the pyro phosphate
added as the salt to the diet caused no harm to the animals.
Happert8 showed that pyro phosphate added to an eXperimental
diet exerted no abnormal influence on the development
of the rat and on reproduction and rearing of its young.

In view of the limited and inconclusive studies
reported in the literature on the utilization and fate
of pyro phosphates in the animal body it was considered
desirable to do further work to determine whether ingested

pyro phOSphate is converted to and used as ortho phOSphate.

EXPERIMENTAL PART I

In order to demonstrate the conversion of pyro
phosphate to ortho phosphate a comparison was made of
the relative effects of equivalent amounts of the basic
and acid pyro and ortho phOSphates when added to a
ricketogenic diet. The use of such a diet in the
production of eXperimental rickets is based on the fact
when the phosphorus of the diet is limited in amount and
restricted to cereal sources in which much of the phosphorus
is in the form of phytin, the addition of a large amount
of calcium carbonate will so decrease utilization of
phosphorus that calcification of the skeleton is
practically entirely inhibited. Blood phosphorus values
drop to an exceedingly low level. If to such a diet
available phosphorus such as sodium ortho phosphate is
added, there is an immediate response in blood phosphorus
values and in bone development.

The method employed therefore was simply to compare
the effects.of equivalent amounts of the basic and acid
salts of pyro and ortho phosphoric acid when supplemented
to a basal ricketogenic diet and fed to young growing rats.

Young rats, 21 to 25 days old and weighing between
AS and 50 grams, were selected and divided into groups
as whown in Table I. The basal diet used in these studies

had the following composition:

Ground yellow corn -------- 1605 grams

Ground oatmeal - ----------- 600 grams
Wheat gluten -------------- 600 grams
Yeast - -------------------- 125 grams
Calcium carbonate --- ------ 90 grams
Sodium chloride ----------- 30 grams

The animals were fed their respective diets for a
period of seven weeks. it the end of this period they
were killed, blood samples collected, and the femurs
removed.

The blood phosphorus value of each animal was
determined by the Youngberg procedure using the Fisher
ElectrOphotometer in place of the calorimeter. The
percent bone ash of the femur of each animal was
determined in the conventional manner, 1.6., on the fat
extracted dessicated bones. The analytical results
obtained in this study are summarized in Table II. The

detailed data are assembled at the end of the thesis.

DATA:

TABLE I

Supplements to the Ricketogenic Diet

 

Amount P contributed
Group No. No. of Animals Supplement by supplement per
100 gm. of ration

 

1 5 none ---

2 6 naco3 reduced to 1% --—

3 6 HaEPah 112320 16h.mg.
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5 6 RaSPOh x 12320 hl mg.
6 6 NsuPZOT x 10320 16n.mg.
7 6 NauPZO7 x IOHZO 82 mg.
8 6 HauEZOT x 10320 hi mg.
9 6 RaHZPOh x.H20 2A6 mg.
10 1h NaHZPOu x 320 16h mg.
11 8 HaHZPOh x H20 82 mg.
12 7 Hafiz?°u,‘ 320 hi mg.
15 7 NaZHZPZOT 1 H20 2&6 mg.
in 7 Na K P 0 x H 0 16h mg.

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Discussion:

in examination of Table II discloses in general that
all additions of ortho or pyro phosphates caused a definite
increase in bone ash, blood phosphorus, and the weight of
the animal. Group 2.was included to demonstrate the effect
of lowering the calcium carbonate content of the diet.

By comparing the average results of the alkaline
salts with the basal diet it may be readily observed that
the addition of increasing amounts of either the tetra
sodium pyro phosphate or the tri sodium ortho phOSphatB
caused a definite improvement in bone deveIOpment as
indicated by the increases in percent bone ash. As might
be expected the blood phosphorus values showed corresponding
increases.

Comparison of the results obtained by feeding the tri
sodium ortho phosphate with those of the tetra sodium pyro
phosphate indicate that the alkaline salts of the pyro
phosphate are not utilized as well as the comparable ortho
phosphates. This conclusion is based on the fact that the
bone ash and the blood phosphorus values were consistently
lower in the case of the tetra sodium pyro phosphate.
Nevertheless, the tetra sodium pyro phosphate was appreciably
utilized in every instance as shown by the increased bone
ash as well as the blood phosphorus values when compared
With the basal group. In contrast the test with the acid

salts reveal striking similarity in effects at all levels.

This indicates the complete utilization and conversion of
the acid pyro phosphate to the ortho phosphate form.
Inasmuch as the acid pyro phosphate employed in baking is
the disodium dihydrogen pyro phosphate and reacts with
sodium bicarbonate to form predominately the trisodium
monohydrogen phosphate, one may assume that the responses
with the latter would at least fall between the limits
extablished for the tetra and the disodium pyro phosphate.
It would accordingly be reasonable to conclude that the
use of disodium dihydrogen pyro phOSphate in baking
contributes appreciably to the amount of useful phosphorus
in a food prepared with an acid pyro phosphate leavening
agent.

On the basis of these results one is permitted to
conclude that the pyro phosphates are well utilized for
maintaining a normal blood phosphorus level and supporting
bone development. It is reasonable to assume that all of
the other uses for phosphorus may likewise be served by

ingested pyro phosphates.

IO

EXPERIMENTAL PART II

Since it was found (Part I) that the pyro phosphate
can be well utilized in bone deve10pment and claims in
the literature have been made that the pyro phosphate is
converted to the ortho phosphate before it reaches the
blood, it was desired to determine whether the method
employed for the analysis of blood was reasonably specific
for ortho phosphate. Accordingly solutions of both ortho
and pyro acid phosphate containing .1 mg. of P per ml.
were made and the phosphorus content of each solution was
determined according to the Youngberg procedure for blood

phOSphorus. The following results were obtained:

Phosphorus Source Actual phosphorus Analyzed P
content content
Socium acid ortho phosphate 8 mg./lOO ml. 8 mg./100 ml.
Sodium acid pyro phosphate 8 mg./100 m1. .6 mg./1oo ml.
Sodium acid pyro phosphate 12 mg./100 ml. .7 mg./100 ml.

Erom the above it is readily observed that the reaction
between pyro phOSphate and the Ibungberg reagent is very
slight.

The small amount determined in the analysis of the
pyro phosphate solution may be due to the conversion of
the pyro to the ortho form by hydrolysis. Upon analysis
of the above solutions at later dates it was found that
the amount of phosphorus gradually increased. At the end

of ten days there was about fifty percent conversion to

the ortho form.

11

It was desired, also, to determine if the trichlor-
acetic acid used in precipitating the proteins in this
method hastened the conversion of the pyro to the ortho

form. The following shows the results obtained:

S.A.P.P. T.C.A.A. Analyzed P Analyzed P
(1 ml. H. 1 mg. P) added content content
(1 hr. after (h days after.
mixing) mixing)
10 ml. 80 ml. .8 mg./lOO ml. 2.9 mg./100 ml.
10 m1. hO ml. .8 mg./IOO ml. 2.8 mg./100 ml.
10 ml. 20 m1. .8 mg./100 m1. 2.76mg./100 ml.
10 ml. 10 ml. .8 mg./lOO ml. 2.7 mg./100 ml.
10 ml. 5 m1. .8 mg./100 ml. 2.61 mg/100 ml.
10 ml. none .8 mg./100 ml. 2.59 mg/100 ml.

The above would indicate that the trichloracetic acid
used in the analysis had little effect in increasing the
conversion of the pyro to the ortho form.

A study now was made of the analysis of blood samples
to which known amounts of ortho and pyro phosphates were

added. The results of these tests follow:

Analysis
Amount and Source of P. Gm. phosphorus [100 ml.
2 ml. water - 5 ml. of blood h.5
2 ml. ortho (1 ml. .1 mg.) - 5 ml. blood 6.h
2 ml. pyro (1 ml. .1 mg.) - 5 ml. blood h.65

It is apparent from the above that there is only a

slight increase in the phOSphorus value when the pyro

12

phosphate is added to the blood sample. This increase
corresponds to that observed when a solution of the pyro
phosphate was analyzed directly. It may be stated then
that the blood values obtained in the main portion of this
study represent ortho phosphate. It also indicates that
ingested pyro phOSphates are converted to the ortho form
before passing into the general circulation.

In order to determine the site of this conversion it
would probably be necessary to examine the blood passing‘
from the digestive tract to the liver. In view of the
marked phosphatase activity of intestinal tissue it would
be reasonable to expect that the change to the ortho form

might occur during the process of assimilation.

2.

13

SUMMARY

Tetra sodium pyro phosphate is not quite as well
utilized for bone deve10pment as tri sodium ortho
phosphate.

The acid pyro phosphate is utilized to the same
extent as the acid ortho phOSphate.

The pyro phosphates are converted to ortho phosphates
before reaching the general circulation.

Essentially only ortho phosphates are determined in

blood when using the Youngberg procedure.

h.

1h

BIBLIOGRAPHY

Gamgee, Priestly and Larmuth, On the difference in
the poisonous activity of phosphorus in the ortho,
meta, and pyro phOSphoric acids., J. of Anat., 11,
255-28h (1877).

Gamel, Schweig, fischr. f. Chem. u. Pharmaz., 39 502,
(1901).

Gardner, J. A., and Symes, W. L., The physiological
action of some camphene phOSphonates of sodium, Bioc.
J., 5, 390‘399 (1910).

Crawford, Alvert 0., A poisonous principle in certain
cotton seed meals., J. of Pharm. and Exp. Therap., l,
519~5h8. (1910)»

Lohman, Biochem. 1., 205, 208, (1928).

Starkenstein., Ebenda, 32, 257, (1911}.

Behrens, B., and Seelkoph, K., On the pharmacology

of the meta-phosphoric acids., Arch. Exp. Pathogie
and Pharm., 169, 258-2h5, (1953).

Hoppert, c. A., A study of the possible toxic effects
of certain compounds that are or may be used in baking

powders., M. S. 6., (1929).

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