University of Southern California Dissertations and Theses

Avocado oil as the source of vitamin D

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Avocado oil as the source of vitamin D

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Content AVOCADO OIL
AS THE SOURCE OP
VITAMIN D
A THESIS PRESENTED TO
THE DEPARTMENT OP CHEMISTRY
UNIVERSITY OP SOUTHERN CALIFORNIA
IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE
DEGREE OF MASTER OF SCIENCE
By
Noble SV Johnson r
June, 1934
UMI Number: EP41454
All rights reserved
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Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author.
Qissertaitofi RffiSsMng
UMI EP41454
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This thesis, w ritte n under the direction of the
candidate’s F aculty Com m ittee and approved by
a ll its members, has been presented to and ac
cepted by the C ouncil on Graduate Study and
Research in p a rtia l fu lfillm e n t of the require
ments fo r the degree of
£
Master of Science
ary
D ean
D ate. . A l t A . A
F a cu lty C om m ittee /
TABLE OF CONTENTS
CHAPTER PAGE
I. INTRODUCTION...................... 1
II. ' HISTORY OF RICKETS ................................. 2
III. PATHOLOGY OF RICKETS ...... ..................... 5
IV. PREVENTION AND CURE OF RICKETS........... 7
V. NATURE OF VITAMIN D.............................. 12
VI. EXPERIMENTAL ........... 16
Extraction of avocado oil. ........... 16
Preparation of unsaponifiable matter of avocado
oil by saponification of the o i l......... 16
Preparation of unsaponifiable matter of avocado
oil by the' continuous alcoholic extraction method 19
Crystalization of phytosterol by acetic anhydride. 20
Test for sterols ............... 23
Tests for phytosterol. ........... .. . 23
Results of identification............... 24
Preparation of irradiated products ......... . . . 24
The "Line Test" for rickets. . . . . . . . . . . . 25
Interpretation of the tests in the gradual curing
of rickets . . . . . . . . . . . . ............. 26
Procedure for "Line Test”........... 26
Feeding and care of animals...................... . 27
Feeding outline............. 28
Diets........................................ 29
Stock diet ................................. 30
CHAPTER PAGE
Steenbock ricket-producing diet, No. 2965 .......... 30
VII. INTERPRETATION AND SUMMARY............................. 60
Interpretion of results ............... 60
Summary............................................... 60
BIBLIOGRAPHY............................................ 68
LIST OF TABLES
TABLE PAGE
I. Determination of Maximum Doseage of Unrefined
Pressed Avocado Oil. ............... . . . . . . . . 31
II. Determination of Maximum Doseage of Unrefined
Pressed Avocado oil. ...... ........... .... 33
III. Curative Diet - Cod Liver Oil (0.2 cc.)............. 35
IV. Curative Diet - Unrefined Pressed Avocado Oil
(0.25 cc.)............................ 3?
V. Curative Diet - Unrefined Pressed Avocado Oil
(0.5 cc.). . .................... 39
VI. Curative Diet - Irradiated Unrefined Pressed Avocado
Oil (0.2 cc.)............... 41
VII. Curative Diet - Irradiated Unrefined Pressed Avocado
Oil (0.5. cc.). ............. .. . . ........... 43
VIII. Curative Diet - Irradiated Unsaponifiable Matter of
Avocado Oil (10 mg.) ............................. 45
IX. Curative Diet - Irradiated Unsaponifiable Matter of
Avocado Oil (20 mg^) ............. 47
X. Curative Diet - Irradiated Phytosterol Acetate
(0.5 mg,)............................................. 49
XI. Curative Diet - Irradiated Phytosterol Acetate
(1 mg.). . . ........................ 51
XII., Curative Diet - Refined Avocado Oil #36 (0.2 cc.). . 53
XIII. Curative Diet - Refined Avocado Oil #37 (0.2 cc.). . 55
XIV. Curative Series ----- Autopsy Results. . 58
LIST OF CHARTS
CHART PAGE
I. Determination of Maximum Doseage of Unrefined
Pressed Avocado Oil................................. 32
II. Determination of Maximum Doseage of Unrefined
Pressed Avocado Oil. ..... .................... 34
III. Curative Diet - Cod Liver Oil (0.2 cc.)........ . . 36
IV. Curative Diet - Unrefined Pressed Avocado Oil
(0.25 cc.)............................ 38
V. Curative Diet - Unrefined Pressed Avocado Oil
(0.5 cc.)............................................. 40
VI. Curative Diet - Irradiated Unrefined Pressed Avocado
Oil (0.2 cc.)............... 42
VII. Curative Diet - Irradiated Unrefined Pressed Avocado
Oil (0.5 cc.). .................... 44
VIII. Curative Diet - Irradiated Unsaponifiable Matter of
Avocado Oil (10 ©g. )............................... 46
IX. Curative Diet - Irradiated Unsaponifiable Matter of
Avocado Oil (20 mg.)........... 48
X. Curative Diet - Irradiated Phytosterol Acetate
(0.5 mg.)............................................. 50
XI. Curative Diet - Irradiated Phytosterol Acetate
(1 mg,). ............................ 52
XII. Curative Diet - Refined Avocado Oil #36 (0.2 cc.). . 54
XIII. Curative Diet - Refined Avocado Oil #37 (0.2 cc.). . 56
XIV. Gain in Weight on Curative Diet - Average of Each
Group. .......................... 57
CHAPTER I
INTRODUCTION
Much work has been done in this laboratory on the
Vitamin Content of Avocados.
The purpose of this investigation was to determine
the Vitamin D content of Avocado oil, irradiated Avocado
oil, and irradiated Phytosterol separated from Avocado oil.
The preparation of Avocado oil is being investigated
in the laboratory, and it is desirable to ascertain whether
or not the Vitamin D potency of the avocado is retained in
the oil, or whether it might be added by irradiation. If
the oil is shown to be a source of Vitamin D, it should be
of value in the prevention or in the cure of rickets.
In order to appreciate the causes and nature of
rickets, a brief discussion of this will be given before the
description of the experimental work.
Vitamin A
Vitamin B
Vitamin C
Vitamin D
Vitamin E
Masters Thesis
Masters
Masters
Masters
Masters
Thesis
Thesis
Thesis
Thesis
Ruth Watson
Ellsworth Youzt
Eugene Waterman
Mildred Ratliff
Emma K. Price
Florence Schelly
CHAPTER II
HISTORY OF RICKETS
Rickets, while probably not so prevalent as formerly,
is still the most common disease of infants in the temperate
zone.-I- McCollum and Simmonds believe it is becoming more
prevalent. It is common in Europe and the United States
and is increasing in Japan and China. It rarely occurs in
Africa, Greenland, and Scandinavia. However, no climate
confers absolute immunity.
It is believed by some that most babies have rickets
to a slight extent, especially in winter.
Outhouse, Macy, and Brekke claim to have proven by
experiments on rats that human milk contains no demonstrable
antirachitic factor and that cow*s milk does.^ Clinical
experience shows that neither is always protective to babies
against rickets.
It has long been believed that sunlight and codliver
oil are of value in curing rickets. Raczynski in 1912 was
Alfred F. Hess, The Contribution of Biology. Chem
istry. and Physics to the Newer Knowledge of Rickets. (Science
LXVII, 1735, March, 1928J.
P
Outhouse, Macy, and Brekke, A Quantitative Com
parison of the Antirachitic Factor in Human Milk and Cow* s
Milk. (J. Biol. Chem., LXVIII, June, 1988'),' p. 129.
3
the first to prove that sunlight plays an important part in
prevention and the cure of rickets.3 In 1919 when
Huldschinsky discovered that ultra-violet rays cure rickets,
a natural conclusion was that ultra-violet rays of sunlight
are the curative agent. Ultra-violet rays pass through
quartz, hut not through ordinary glass. If an infant cannot
he exposed to direct sunshine, it may be treated with ultra
violet light from a mercury vapor lamp, or receive sunlight
through a special kind of a glass such as Vitoglass, which
transmits 85 to 50 per cent of the ultra-violet rays.
The feeding of irradiated brain is being used to cure
rickets, as it contains more cholesterol than any other
organ.
Independently, but almost simultaneously, in 1925,
three groups of workers, Rosenheim and Webster,4 Steenboch
and Black,5 and Hess, Weihstock, and Helman,6 discovered
3 McCollum and Simmonds, The Newer Knowledge of
Nutrition. (Third Edition, 1927). p. 430
4 Rosenheim and Webster, Rickets and Cholesterol.
(Lancat I, May 1925). p. 1025.
Steenboch and Black, Induction of Growth Promoting
and Calcifying Properties in Fats and their Unsaponifiable
Constituents by Exposure to Light. TJ. Biol. Chem., LXIV,
June, 1925). p. 1263.
3 Hess, Weinstock, and Helman, Antirachitic Value
of Irradiated Phytosterol and Cholesterol. (J. Biol. Chem.,
LXIII, March, 1925). p. 305.
that certain sterols, or certain foods containing
cholesterol or an allied substance when irradiated with
ultra-violet rays would cure rickets. Vegetable oils,
cereals, and dried milk being quite valuable for treatment
CHAPTER III
PATHOLOGY OF RICKETS
The essential feature of rickets is the failure of
the bones to become calcified, which causes them to remain
so soft that they are easily bent out of shapiand deformities
result. Due to faulty metabolism of calcium and phosphorus,
or dificiency of either or both in the diet, there is a low
concentration of one or of both of these elements in the
blood or a faulty ratio of the two, which results in
insufficient calcium being deposited in the bone.
There are three types of rickets: (1) low-phosphorus
rickets, when the calcium content of the serum is normal,
but the concentration of the phosphorus is reduced. This is
the common rickets found in infants and easily produced in
experimental animals. The ends of the long bones become
enlarged because of an overgrowth of cartilaginous or
osteoid tissue and because of the pressure of the body
weight on the soft part, A rachitic rosary, or line of knobs,
appears on the. ribs and the thorax becomes deformed: (2) low-
calcium rickets, with the phosphorus content of the serum
normal, but with the calcium content reduced. This results
in somewhat similar abnormalities, but is often accompanied
by tetany^ the lack of calcium in the blood affecting the nerves
as well as the bones: (3) osteoporosis, differing in clinical
6
and histological appearance. This results from a reduction
of both calcium and phosphorus in the blood. Of these three
types, the low-phosphorus type is the one usually studied.■ * -
- * ■ Henry G. Sherman, Chemistry of Food and Nutrition,
(Third Edition, 1927). p. 474.
CHAPTER IV
PREVENTION AND CURE OF RICKETS
Antirachitic refers to the prevention or cure of
low-phosphorus rickets in children.or experimental animals.
It is suspected at the very beginning that rickets
was caused by the lack of some particular substance in
one's diet. Various experiments have been undertaken to
discover a diet which would produce rickets in animals.
McCollum and Simmonds, 1917-1919, working with rats, and
Meldanby, 1919, working with dogs, succeeded in producing
rickets. It has been demonstrated by many workers that
rickets may be caused in one of three ways or by a
combination of these; (l) deficiency of the anti-rachitic
vitamin, (2) reduction of the amount of calcium or
phosphorus in the blood or a change in the ratio, (3)
o
deficiency of the ultra-violet irradiation.
Meldanby was the first to associate fat-soluble
vitamin with the prevention of rickets. Huldschinsky, 1919,
discovered that ultra-violet rays prevent or cure rickets.3
McCollum and Simmonds, The Newer Knowledge of
Nutrition, (Third Edition, 1927). pp. 374-375.
^ Henry C. Sherman, Chemistry of Food and Nutrition.
(Third Edition, 1927). p. 474.
3 McCollum and Simmonds, The Newer Knowledge of
Nutrition. (Third Edition, 1927). pp. 374-375.
8
These factors led to the newer methods in the study and cure
of rickets.
Very little was known about vitamins before 1920. The
use of animals for nutritional experimentation has led to
their discovery and differentiation. Vitamins are named
according to the dificiency-disease they prevent.
Vitamin D is the most poorly distributed among foods
of all vitamins. It has not been identified in cereals of
fruits and is absent or negligible in vegetables.4 Nursing
mothers and infants have their most available source in egg
yolk, liver, and to a less extent in some of the animal fats.
Cod liver oil is one of the most abundant sources of vitamin
D. Jones, Murphy, and Nelson recently have found 65 mg. of
oyster fat antirachitically equivalent to 4 mg. of cod liver
oil.5 They found considerable more vitamin D in clams.®
Hess and Weinstock think that the small amount of antirachitic
factor associated with the fat of cow’s milk is more than
counterbalanced by the constituents in the remaining portion;
cow’s milk, therefore, being productive of, rathfer than a cure
4 Hess and Weinstock, A_ Study of the Antirachitic
Factor in Human and in Cow’s Milk. (Am. F. Dis. Child, 'XXXIV,
November 1927). pp. 845-853.
5 Jones, Murphy, and Nelson, Vitamins in Oysters. (Ind.
and Eng. Chem., XX, February 1928). pp. 205-210.
6 Jones, Murphy, and Nelson, Vitamins in Clams. (Ibid.,
XX, June 1928). pp. 648-652.
for rickets. Human milk contains only a small amount of
vitamin D.^
Subcutaneous injection of codliver oil or of irradiated
cholesterol dissolved in ether mall cure rickets.® It has
also been shown that, when irradiated cholesterol is applied
to a small area of the skin, enough is absorbed to cure
rickets.8
The skin itself contains cholesterol or a similar agent,
since it has been found that rats fed irradiated portions of
skin are cured of rickets.^® The sterols must be superficial
to the pigment layer, as Levinsohn has shown that the pigmented
skin of the negro does not retard the effect of ultra-violet
rays in curing rickets.
Vitamin D may, therefore, reach the blood by absorption
through the skin capillaries from naturally or artificially
7
Hess and Wemstock, A Study of the Antirachitic
Factor in Human and in Cow’s Milk. " (Am. F. Dis. Chils. XXXIV,
November, 1927). pp. 845-853.
8 Kramer, Kramer, Shelling, and Shear, God Liver Oil
Goneentrate. (J. Biol. Chem., LXXI, February 1927). pp. 699-706.
8 Hume, Lucas, and Smith, On the Absorption of Vitamin
D from the Skin. ( Biochem J., XXI, 1927). pp. 362-367.
10 Rosenheim and Webster, Photochencial Production of
Vitamin D from Ergosterol. (Lancet, II, September 1927). pp.
622-625..
■ j * 1
Levinsohn, Rickets in the Negro. (Am. J. Dis. Child.,
XXXIV, December 1927). pp. 955-961.
10
irradiated sterols. The vitamin may also reach the blood by
absorption from the intestine from substances naturally
containing it, such as cod liver oil, or from irradiated
sterols or irradiated foods containing sterols. Vitamin D,,
thus reaching the blood from the skin or intestine, enables
the calcium and phosphorus in the body to be utilized in
bone formation.
Raczynski in 1915 found sunlight favorable to calcium
assimilation in puppies, and definitely suggested a connection
between the absence of sunlight and rickets.4^
Huldschinsky in 1919 and 1980 reported that ultra
violet light cured children of rickets.-*'®
Hess and Unger in 1921 confirmed Huldschinsky*s reports
by showing that exposure to ultra-violet light or sunshine,
without any modification of the diet would cure infantile
rickets.
Hess and Lundagen in 1922 found a seasonal tide of
blood phosphate which could be correlated with the seasonal
variation of ultra-violet light waves in the sun’s spectrum.-*-®
^ Sherman and Smith, The Vitamins. (Second Edition),
pp, 293-305.
15 Ibid.
14 Ibid.
4® Ibid.
11
Hess, Pappenheimer and Weinstock in 1922 showed that
light waves longer than 334 u.u. are ineffective while shorter
than 310 u.u. are effective in preventing rickets in rats on
a drastically rachitic diet.-^
The above reports indicate that vitamin D or ultra
violet light can help an organism to utilize advantageously
the available calcium or phosphorus, but cannot replace an
actual deficiency.
16 Ibid.
CHAPTER V
NATURE OF VITAMIN D
In August 1922, McCollum and his co-workers furnished
explicit evidence of the existence of vitamin D, as an
entity distinct from Vitamin A. In summarizing their
results, they stated that "cod liver oil oxidized from 10 to
12 hours does not cure xerophthalmia in rats." It does,
however, cause the deposition of calcium in the hones of
young rats which are suffering from rickets. This shows that
oxidation destroys fat-soluable A without destroying another
substance which plays an important role in bone gro?rth.^
In 1923 Steinbock and Nelson confirmed McCollumTs
experiment.^
Dubin and Funk in 1923 found vitamin D stable to
hydrogenation at 55° C. for 36 hours with colloidal platinum
as a catalyst.^
Bill’s recorded in 1925 that it is not destroyed by
hydrogen dioxide, hydrogen sulphide, sulfur dioxide or
formaldehyde, but is readily destroyed by nitrous fumes and
Sherman and Smith, The Vitamins, (Second Edition).
pp. 293-335.
2 Ibid.
3 Ibid.
13
slowly by direct steam or contact with mineral acids.4
Hess, Weinstock, and Sherman in 1986 succeeded in
separating irradiated cholesterol into an active and inactive
portion by precipitating the inactive cholesterol with
digitonin. The active fraction constituted 4 or 5 per cent of
the original.^
Rosenheim and Webster in 1927 found both the unsaturated
carbon linkage and the secondary alcohol grouping essential
to the precursor of vitamin D.6
Hess and Windous in 1926-1927 published evidence that
cholesterol or phytosterol, recovered after being separated
twice as the dibromide, could not be activated. They
questioned whether in previous work, cholesterol or a con
taminating substance had been activated. Soon afterward they
reported that ergosterol, a substance having 3 double bonds
and a hydroxyl group, could be activated by irradiation.7
Rosenheim and Webster, after their work with the
spectroscope, also conclude that the naturally occurring
parent substance of vitamin D is ergosterol or a sterol highly
4 Ibid.
5 Ibid.
6 Ibid.
7 Ibid.
14
-unsaturated and of similar constitution possessing the same
absorption spectrum and physiological activity.®
Kock, Rogins, and Lemon in 1929 believed that the pro
vitamin D activity may be a general property in varying
degrees of various sterols or of certain forms of these
sterols.9
However, various other experimentors believe, if
ergosterol is the sole antirachitic precursor, it is evident
that this sterol must be universally present in all fats of
animal and of plant origin that are capable of activation by
ultra-violet radions.-*-9
The wave-lengths which vitamin D itself absorbs, and
by which it is destroyed, apparently lie within the same
spectral region as the wave-lengths which activate ergosterol.
Reering and Van Vijk. Irradiation of ergosterol with
light of wave-length 254 u.u. (nshort-wave” irradiation) gives
rise to a series of reaction products different from that
caused by irradiation with light of wave-length 275 u.u.
("long-wave” irradiation)
Zucker, Pappenheimer, and Barnett in 1922 showed the
8 Ibid.
9 Ibid.
10 Ibid.
11 Ibid.
15
stability of vitamin D to saponification.4^
Southgate in 1925 found that the antirachitic vitamin
of cod liver oil was destroyed partially by heating for two
hours at 200 C. in absence of air, and to a still greater
degree by heating for four hours at 200°C.43
Marshal and Khudson in 1930 Summary: The rate of
production of vitamin D from ergosterol is proportional to
the first poy/er of the light intensity. The rate of production
of vitamin D is directly proportional to the number of light
quanta absorbed by ergosterol and independent of the wave
length of the light used.44
12 Ibid.
13 Ibid.
14 Ibid.
CHAPTER VI
EXPERIMENTAL
EXTRACTION OF AVOCADO OIL
The avocado oil was extracted from ripe avocados. The
avocados were first pealed; then cut into very thin slices,
and dried in a dehydrater. The oil from the dried avocados
was extracted by means of a hydro-static press and was then
filtered.^-
PREPARATION OF UNSAPONIFIABLE MATTER OF AVOCADO OIL
BY SAPONIFICATION OF THE OIL
Esters have the known property of being easily
saponified by strong alkalis, producing a soap
and the free glycerol. Soap and glycerol are
soluble in water while the sterols are not; this
property being made use of in the extraction
and isolation of sterols.
Apparatus: 1000 cc, 250 cc. Erlennieyer flasks, reflux
condenser, thermometer, water bath, 1000 cc.
separatory funnel.
The extraction of the oil was done by Mr. Russell
Sanborn in his research work on preparing avocado oils.
17
Chemicals: Con. KOH.-1.000 g/liter
Dll. KOH.-11.2 g/liter
Phenolphthalein.-Dissolve one gram of
phenolphthalein in 100 cc. alcohol.
Ethyl alcohol.-approx. 95$ by volume
Ether.- U.S.P. grade
Method: Accurately weigh 100 grams of the avocado
oil into a 1000 cc. Erlenmeyer flask. Add 250 cc.
alcohol and 100 cc. of the concentrated alkali
solution. Mix thoroughly by rotating flask.
Boil the mixture, using the reflux condenser for
twenty minutes. Cool to 30° C., add 250 cc. of
ether, mix, and carefully transfer to a 1000 cc.
separatory funnel. Rinse the flask with two
successive 50 cc. portions of ether, add to the
separatory funnel, and mix the contents thoroughly,
wash the saponfication flask with 100 cc. of the
dilute alkali solution and pour into the separatory
funnel in a slow steady stream. Rotate the funnel
gently to secure better contact of the solutions,
but do not shake. Shaking at this stage causes
stubborn emulsions. Allow the liquids to
separate completely, and then draw off as much of
the soap solution as possible, slowly. Do not
draw off any of the emulsion that may be formed.
18
Keep the volume of ether at about S50 cc. by
replacing that dissolved by the wash solutions.
Further treat the ether solution with two
successive 100 cc. portions of the wash solution
in the same manner as described above. Add 50 cc.
of water to the ether solution and mix by rapid
rotation of the separatory funnel. When the
layers have separated withdraw the water. Repeat
this treatment by shaking the water and ether
thoroughly until the washings are free from
alkali, as shown by testing with phenolphthalein
solution. Three washings usually suffice. Transfer
the ether quantitatively to a weighed 250 cc.
Erlenmeyer flask. Distill the ether, using a
water bath, then dry the flask and residue in an
O °
oven heated to 100 C. or 105 C., until a
constant wreight is obtained. From this the per
centage of unsaponifiable matter can be calculated.
Comment: In connection with the distillation of the
ether, it is preferable to employ a suitable spray
trap betv/een the flask and the condenser to
eliminate any possible loss of unsaponifiable
matter. It will be observed that this method
in contrast to others is based upon the solution
of the unsaponifiable matter in ether and the removal
of dissolved soap by washing with a dilute
solution of potassium hydroxide, which is
necessa.ry to prevent the formation of acid soaps
which cannot be removed from the ether.
PREPARATION OF UNSAPONIFIABLE MATTER OF AVOCADO OIL
BY THE CONTINUOUS ALCOHOLIC EXTRACTION METHOD
Apparatus: 700 cc. round-bottom flask, reflux con
denser, 1000 cc. Erlenmeyer flask, 1000 cc.
separatory funnel.
Chemicals: Ethyl alcohol, ether U.S.P., phenolphthalein,
concentrated potassium hydroxide.
Method:- Accurately weight 100 grams of the avocado oil
into the round-bottom flask. Place 700 cc.
Aldehyde free 95$ ethyl alcohol in Erlenmeyer
flask. Keep both flasks in a warm water bath.
Heat the alcohol in the Erlenmener flask. The
alcohol distilled through the warm oil refluxes and
collects in a layer on top. Continue the process
for ten hours. Transfer contents of both flasks
to a separatory funnel. Rinse both flasks with
two successive portions of alcohol, add to the
separatory funnel. Allow the liquids to separate
completely, and then draw off the oil and transfer
the alcohol to an Erlenmeyer flask. Add 40 cc.
. 20
1:1 potassium hydroxide solution, boil 10 minutes;
add to 500 cc. warm ether. Transfer to separatory
funnel-mix. (Do not form emulsion). Add 50 cc.
of water to the ether solution and mix by rapid
rotation of the separatory funnel. When the
layers have separated withdraw alcohol water soap
solution. Repeat this treatment by shaking the
water and ether thoroughly until the washings are
free from alkali, as shown by testing with
phenolphthalein solution. Transfer the ether
quantitatively to a weighed 250 cc. Erlenmeyer
flask. Distill the ether, using water bath, then
dry the flask and residue in an oven heated to
0 0
100 C. to 105 C. until a constant weight is
obtained. Calculate percentage of unsaponifiable
matter.
Results: Percentage of unsaponifiable matter by
saponification of oil.......... ... .5.3095g/100g oil
Percentage of unsaponifiable matter by the
continuous extracting of the unsaponifiable
matter with warm alcohol....... , ,4.5528g/100g oil
CRYSTALIZATION OF PHYT0STER0L BY ACETIC ANHYDRIDE
Transfer the unsaponifiable matter to a 50 cc. Erlenmeyer
flask and add about 10 cc. of acetic anhydride. Insert a
ttjrrttntti uo run rn a rjiiiA ixu a ur u iw a ru u iiriiiriiti ia.tti.xnri ur
AVOCADO OIL BY THE CONTINUOUS ALCOHOLIC EXTRACTION METHOD
SI
<-Condenser
v' \\\
V\ \
\\\ \\
f i t / j
Erlenmeyer
<— flask
Round-bottom
flask
Electric
<— Heater
-000000 0 Q JJt 0 0 O 0 O 0 j 0JL0Jf £i
22
crucible cover over the neck of the flask and boil the
contents gently for about 30 minutes while, the flask rests
on an asbestos-wire gauze; when cold, add 30 to 35 cc. of
alcohol, 60 per cent by volume, and mix thoroughly. Filter
and wash the precipitate four or five times with the 60 per
cent alcohol; then dissolve it on the filter with a fine
stream from a wash bottle of hot alcohol, 80 per cent by
volume, Place the filtrate in the ice-box for several hours,
0
or m a water bath maintained at about 10 C. Collect the
crystalline acetates upon a filter, wash with cold 80 per
cent alcohol, and then dissolve in a minimum quantity of
hot absolute alcohol, receiving the filtrate in a small
beaker. Add 2 drops of water and warm if not perfectly clear.
0
Place in ice box at 10 C. Allow the alcohol to evaporate
spontaneously, stirring the contents occassionally to mix
the deposit of crystals that form ujjon the edges with the
main body of the liquid. As soon as a good deposit of
crystals have formed, filter them upon a hardened filter, wash
twice with cold alcohol, 90 per cent by volume, and dry them
at 100° C. for 30 minutes. Then determine the melting point.
The melting point of cholesteryl acetate is 114° C. and that
< * S >
of phytosteryl acetate is usually from 125 to 138 C. The
melting point of the first crop of crystals usually.:gives
definite information as to the presence or absence of
phytosterol, but the conclusion indicated should be confirmed
23
by recrystallizing the crystals from absolute alcohol and
again determining the melting point.
TEST FOR STEROLS
LIEBERMAN’S TEST: A small quantity of the sterol or
unsaponifiable matter separated from a fat is dissolved in
2 cc. of acetic anhydride and on adding sulfuric acid (3 to
5 drops) a violet pink color is developed.
BURCHARD’S MODIFICATION; Dissolve the samples to be
tested in 2 cc. of chloroform, 20 drops of acetic anhydride
and 2 drops of sulphuric acid. The violet-reddish colors
given soon change to blue or green. On account of the
violet-reddish colors given by resins, care should be taken
that none of these substances are present when the sterol
tests are made.
TESTS FOR PHIT0STER0L
Cholesterol and phytosterol are derivatives of the
phemantrene group, having the same groups in their side
chains and being isomeres; so when treated with acetic
anhydride and sulphuric acid, they produce condensation
reactions, and give the same color reactions. Cholesterol
crystals are of a very wide shape, phytosterol crystals are
needles of romboidal form or narrow plates.2 These crystals
2 Leach, Food Inspection and Analysis, (1920), p. 524.
24
sometimes are joined forming a characteristic star.
RESULTS OF IDENTIFICATION
Sterols: Positive reactions were given in testing the un
saponifiable matter, by both methods of extraction, for sterols
by the Lieberman’s Test and Burchard’s modification.
Phytosterol: Microscopic examination showed fine needle
shaped crystals of phytosterol acetate having a melting point
O
of 129.1 C. (cor) The crystals formed the characteristic
star formation.
PREPARATION OF IRRADIATED PRODUCTS
Irradiation of avocado oil. Avocaco oil was spread in
a thin film, approximately 1 mm. deep, on a round 18 cm. flat-
bottom glass dish. This ?/as irradiated with ultra-violet
light for 20 minutes at a distance of 30 cm.3
Irradiation of the unsaponifiable matter. Two grams
of the unsaponifiable matter were dissolved in 10 cc. of
peanut oil and then warmed on a water bath to facilitate the
pouring of the mixture on to the flat-bottom glass dish.4
Lamp used. Cooper Hewith Mercury Vapor Quartz Lamp.
4 Peanut oil does not contain any antirachitic
properties.
25
TMs was Irradiated with ultra-violet light for 20 minutes
at a distance of 50 cm.
Irradiation of phytosterol acetate. 50 mg. of
phytosterol acetate crystals, purified several times by
dissolving in absolute ethyl alcohol and successive
crystallization was dissolved in 5 cc. of peanut oil. This
was irradiated with ultra-violet light for 20 minutes at a
distance of 30 cm.
All the irradiated products were stored in a,dark glass
bottle.
THE "LINE TEST” FOR RICKETS
The ”Line Test” Is a test for calcium— depositing
antirachitic substances.
The ”Line Test” depends upon the presence of a
provisional zone of calcification in the epiphyseal cartilage,
easily seen in the ends of the tibia and femur of a normal
rat, disappearing in severe rickets, and reappearing in
healing rickets.®
In a "Negative Line Test” there is no calcification
of the epiphyseal cartilage and little if any in the metaphysia
® McCollum and Slmmonds, A Delicate Biological Test
for Calcium-depositing Substances. (J. Biol. Chem., LI,
January 1922) ’ . pp. 41-47.
26
In a "Positive Line Test" there is a broad linear deposit of
calcium salts on the metaphyseal side of the epiphyseal
cartilage,8
INTERPRETATION OF THE TESTS IN THE GRADUAL
CURING OF RICKETS <
0 negative
/.healing is considered a negative test
/ / healing positive test
/ / / healing positive test
/ / / / healing positive test, normal
PROCEDURE FOR "LINE TEST"8
At the conclusion of the tenth day of the experi
mental period, the animals were killed and the tibia was
removed from the right leg and treated as follows:
1. Dissect out tibia, scrape clean.
2. Split bone sagittally
3. Soak in formaldehyde (10%) for 24 hours
4. Rinse in distilled water
8 Sherman and Smith, The Vitamins. (Second Edition).
pp. 314—315
^ E. Bills and F. G. McDonald, (Jour, of B. Chem., 68,
1926). p. 821.
8 Journal of Pharm. Ass. June 1930. p. 623.
27
5. Place in acetone for 5 minutes
6. Rinse in distilled water
7. Dry bone on blotter
8. Place bone in silver nitrate solution (2%) for 3 minutes
9. Intensify under bright light and watch until line is
evident
10. Place in sodium thiosulphate solution (5%) for 3 minutes
11. For keeping, store bones under distilled water. Add a
couple drops of formaldehyde to prevent the growth of
bacteria and molds.
FEEDING AND CARE OF ANIMALS
Each group of rats was placed in a separate cage and
kept in good sanitary condition. Approximately ten grams
of the respective diet was given to each rat daily and
plenty of fresh water. The room in which the rats were kept
was well ventilated- and kept at a reasonable temperature.
In order not to have the rats Irradiated by direct sunlight,
the shades vrere kept pulled down.
The'method of feeding the animals and frequency of
feedings was satisfactory as outlined by the Committee on
Vitamin Research.9 a hypodermic syringe was used instead of
an eye dropper to feed the oil to the animals. This
Q
Journal American Pharm. Ass., June 1930. p. 623.
28
eliminated the old method of determining the volume of so
many drops of oil; also, better measurements could be made
from day to day as the change in viscosity of the oil due to
the change in temperature from day to day would not influence
the readings appreciably.
If the dropping— pans are cleaned each day, one is
not forced to fight ants; as there will be very little food
on the dropping— pans to attract the ants. This same method
of cleanliness was followed even when the mother rat had pups.
T his did not make the mother nervous so that she would
disturb the young, providing it was done gently.
If the room in which the rats are kept is. too dark,
fungus will occur on the ears of the rats. This is quickly
eliminated or cured by giving the animals a little more
light, not direct sunshine.
When the feeding outline so given is followed, there
should be no trouble in reducing the animals to a rachitic
state.
FEEDING OUTLINE
Birth to 10 to 14 days-------------------------- Stock diet
12 to 14 days (22 to 28 days of age)------------ Restricted diet
Wean animals when 28 days old
18 to 21 days feed Steenbock Ricket-Producing Diet, No.2965
After the experimental animals have been on the rachitic
29
diet for a suitable period (18 to 21 days) examine one rat
out of each litter, by the ,!line test11, to see what stage
of rickets has been attained. If these animals are found
to have developed definite rickets, the remainder are con
sidered as satisfactory for the test.
DIETS
The diets had to be adjusted a little from the
diets fed by previous research workers.
The calcium content of the restricted diet was re
duced to the level where the mother rat could tolerate it
in the diet.
Dry yeast was substituted for the irradiated dry
yeast as it took too long to reduce the pups to the rachitic
state.
The maximum doseage of raw avocado oil that a rat
could tolerate was determined. It was found that 0.01 cc. per
gram of body weight was most satisfactory. Any amount over
this acted as a purgative, as the animal’s stools were
watery. The animal naturally lost weight. Large doseages
were not toxic as animals removed from a diet of excessive oil
immediately gained weight when taken off of the diet containing
oil. Or when placed on a diet containing oil of a limited
amount; not only gained weight, but in excess of those rats
on the same diet minus the avocado oil.
so
STOCK DIET
Whole yellow corn meal, ground---------------------- 400 grams
Whole wheat flour 400 grams
Whole milk powder-------------------------------------300 grams
Calcium carbonate--------------------------- : ---■ 5 grams
Sodium chloride 5 grams
Dry yeast 10 grams
Lettuce, daily
RESTRICTED DIET
Whole wheat 2 parts
Skim-milk powder 1 part
Sodium chloride, 1D% of weight of wheat.
Calcium carbonate, 2.5% to 3.0% of weight of wheat.
STEENB0CK RICKET-PRGDUCING DIET, No. 2965
Whole yellow corn (freshly ground) ----------------- 76%
fifheat gluten ------------------------------------------20%
Calcium carbonate ------------------------------------ 3%
Sodium chloride ------------------------- 1%
31
TABLE I
DETERMINATION OF MAXIMUM DOSEAGE OF
UNREFINED PRESSED AYOCADO OIL
Weight in grams
Diet-Stock diet
Doseage lcc. lcc. lcc. lcc. 1.25 1.25cc. 1.25cc
Date 6/30 7/8 7/15 7/22 7/22 7/31 8/5
Rat no. 1 87.4 96.6 112.0 118.0 118.0 110'.0 108.0
Rat no. £ 97.4 108.4 128.5 134.0 134.0
129.0
126.0
Rat no. 3 93.0 113.5- 123.0 128.5 128.5 122.5 120.0
Control 91.0 94.0 , 97.5 101.0 101.0 103.1 105.5

S3
TABLE II
DETERMINATION OF MAXIMUM DOSEAGE OF
UNREFINED PRESSED AVOCADO OIL
Weight in grams
Diet - Stock
Doseage 1.5 1.5 1.5 1.5 1.5 1.5 1.5cc
Date 6/30 7/8 7/15 7/22 7/31 8/3 8/5
Rat no.l 85.1 65.2 83.0 100.0 92.0
91# —
Rat no.S 77.7 61.1
79.0 96.0 90.0 — CO
Rat no.4 78.4 61.2
84.0 97.0
90.0
—
89##
Control 83.0 85.3 89.0 93.0 96.4 99.1
# Died
## Chloroformed them. Too weak to eat.
ffl
M3L
m
35
TABLE III
CURATIVE DIET - COD LIVER OIL (0.2 cc.)
Weight in grams
Oil Added --- Line Test
Date 7/1 7/8 7/31 8/5 8/10
Rat no. 1 23 ;.l 30.0 23.1 28.0 33.0 4
Rat no. 4 25.4 29.3 28.0 32.6 35.9 4
Rat no. 6. 27.6 29.8 26.0 32.0 36.0 4
Control 26.3 29.4 27.1 26.3 26.0 0
B
37
TABLE IV
CURATIVE DIET - UNREFINED PRESSED AVOCADO OIL (0.25 cc.)
Weight in grams
---
Oil Added
---
Line
Date 7/27 8/8 8/15 8/20 8/25
Rat no. 1 21.5 24.0 25.2 26.5 27.5 2
Rat no. 2 21.0 23.1 24.4 25.7 26.7 2
Control 24.0 27.1 28.9 28.0 ro
Co
k:
0
Si
la
i - g f
m
ISHi
39
TABLE V
CURATIVE DIET - UNREFINED PRESSED AVOCADO OIL (0.5 cc.)
Weight in grams
— — Oil Added--------- Line Test
Date 7/1 7/8 7/31 8/5 8/7 8/10
Rat No. 3 26.5 33.5 28.0 32.0 34.2 34.2 2
Rat No. 5 23.0 28.4 21.0 to
«
o
22.0# 2
Control 25.2 29.8 26.4 26.2 26.0 26.1 0
# Died. Too much oil for size of animal.

41
TABLE VI
CURATIVE DIET - IRRADIATED UNREFINED PRESSED
AVOCADO OIL (0.2 cc.)
Weight in grams
---i
Oil Added
---
Line 1
Date 7/1 7/8 7/31 8/5 8/10
Rat no. 1 28.0 54.7 33.5 39.7 45.8 2
Rat no. 2 25.9 31.6 28.0 34.0 39.5 2
Rat no. 5 24.2 27.0 27.0 27.4 29.2 2
Control 24.2 30.0 31.1 30.0 30.1 0

43
TABLE VII
CURATIVE DIET - IRRADIATED UNREFINED- PRESSED
AVOCADO OIL (0.5 cc.) '
Weight in grams
— Oil Added Line Test
Date 7/1 7/8 7/31 8/5 8/10
Rat no. 1 23.1 24.0 21.0 27.0 29.0 3
Rat no. 2 26.6 33.7 31.0 40.0 42.0 3
Control 24.2 30.0 31.1 30.0 30.1 0
m
45
TABLE VIII
CURATIVE DIET -.IRRADIATED UNSAPONIFIABLE MATTER
OF AVOCADO OIL (10 mg.)
Y/eight in grams
Oil Added - Line 1
Date 7/1 7/8 7/31 8/3 8/9 8/10
Rat no. 1 SI. 2 23.8 21.0 27.0 28.6 28.9 1
Rat no. 5 22.9 25.2 27 i'5 31.8 33.0 33.4 1
Rat no. 6 24.2 27.7 30.0 34.0 35.3 35.5 1
Rat no. 10 19.0 22.0 23.5 25.5 31.3 31.9 1
Control 23.0 26.8 26.0 25.8 25.4 25.2 0
The unsaponifiable matter of the avocado oil was
dissolved in peanut oil and then irradiated. 10 mg. of
•irradiated unsaponifiable matter of avocado oil per 0.1 cc.
of peanut oil.
f f l r p
47
TABLE IX
CURATIVE DIET - IRRADIATED UNSAPONIFIABLE MATTER
OF AVOCADO OIL (20 mg.)
Weight in grams
Oil Added Line Test
Date 7/1 7/8 7/31 8/3 8/9 8/10
Rat no. 2 • 24.2 27.0 26.0 30.0 33.5 33.9 3
Rat no. 4 22.5 27.2 26.0 29.0 32.0 32.5 3
Rat no. 5 22.5 24.0 21.5 23.8 27.0 27.6 3
Control 25.0 26.8 26.0 25.8 25.4 25.2 0
The unsaponifiable matter of the avocado oil was
dissolved in peanut oil and then irradiated. 20 mg. of
irradiated unsaponifiable matter of avocado oil per 0.1 cc.
of peanut oil.
. C O ,
m
49
TABLE X
CURATIVE DIET - IRRADIATED PBYTOSTEROL
ACETATE (0.5 mg.)
Weight in grams
Oil Added --- Line Test
Date 7/24 7/51 8/10 8/15 8/20
Rat no. 1 24.2 27.7 50.0 55.0 54.0 1
Rat no. 2 25.0 25.2 29.2 29.2 28.7 1
Rat no. 5 19.1 20.2 21.4 22.7 24.0 1
Rat no. 4 18.2 19.5 20.1 20.7 21.5 1
Control 24.0 27.1 28.2 28.4 27.4 0
^ 0.5 mg. of phytosterol acetate per 0.1 cc. of peanut oil.
HKBiiiKiBSiHBHiiiiiHSiiHiHi
■aiiiiBiiiiiiiii«iPiMi ■■■■■■■■■■»»■«*
51
TABLE XI
CURATIVE DIET - IRRADIATED PHYTOSTEROL
ACETATE1 (1 mg.)
Weight in grams
Oil Added Line Test
Date 7/24 7/31 8/10 8/15 8/20
Rat no. 1 19.1 20.5 22.0 22.3 24.5 2
Rat no. 2 21.0 23.8 23.4 26.1 26.5 2
Rat no. 3 20.8 23.3 24.0 26.3 27.1 2
Control 24.0 27.1 28/2 27.1 27.4 0
1 1.0 mg. of phytosterol acetate per 0.1 cc. of peanut oil.
©I
55
TABLE XII
CURATIVE DIET - REFINED AVOCADO OIL #36 (0.2- cc.)
Weight in grams
Oil Added Line Test
Date 7/24 8/5 8/15 8/20 8/25
Rat no. 1
o
to
02
28.1 29.5 31.1 32.5 2
Rat no. 2 18.0 21.0 20.8 26.0 31.3 2
Rat no. 6 19.8 23.0 23.5 24.4 25.2 2
Control 22.0 25.1 . 26.2 26.0 25.5 0

55
TABLE XIII
CURATIVE DIET - REFINED AVOCADO OIL #37 (0.2 cc.)
Weight in grams
Oil Added Line Test
Date 7/24 8/5 8/15 8/20 8/25
Rat no. 1 34.1 37.0 37.3 37.5 37.7 2
Rat no. 2 36.0 37.8 38.5 40.5 42.3 2
Rat no. 3 31.0 33.0 33.7 33.9 33.0 2
Control 22.0 25.1 26.2 26.0 25.5 0

m
i u a i i
i i u i i
TABLE XIV
58
CURATIVE SERIES ------- AUTOPSY RESULTS
(10 Days on Test Diet)
Diet Rat # Line Test
Rachitic 7 controls 0
Addition of 0.2 cc of cod liver oil 1 4
4 4
,
6 4
Addition of 0,25 cc. of avocado oil 1
3
2 2
Addition of 0.5 cc. of avocado oil 3 2
5 2
Addition of 0.5 cc. of irradiated
avocado oil 2 2
5 2
Addition of 0.5 cc. of irradiated
avocado oil 1 3
2 3
Addition of irradiated unsaponifiable
matter of avocado oil (10 mg.) 1 1
3 1
6 1
10 1
Addition of irradiated unsaponifiable
matter of avocado oil (20 mg.) 2 3
4 3
5 3
Addition of 0.5 mg of irradiated
phytosterol acetate 1 1
2 1
3 1
4 1
59
TABLE XIV (Cont’d)
CURATIVE SERIES ------- AUTOPSY RESULTS
(10 Days on Test Diet)
Diet Rat # Line Test
Addition of 1 mg. of irradiated
phytosterol acetate 1 2
2 2
5 2
Addition of 0.2 cc. of refined
avocado oil #36 1 2
2 2
6 2
Addition of 0.2 cc. of refined'
avocado oil #37 1 2
2 8
3 2
CHAPTER VII
60
INTERPRETATION AND SUMMARY
INTERPRETATION OF RESULTS
In the curative series, the growth curves (Charts I
to XIII, page 31 to 56) show a slight increase in weight
while on the rachitic diet. However, during the last few
days, the increase is not so great, and there is a slight
drop in the weight in most of the groups when the curative
diet was begun. All of the negative controls continued to
lose weight. None of the controls showed healing rickets
by the line test when they lost weight.
Autopsy results for the curative series are shown on
Table XIV, page 58. The line test showed a varying degree
of calcification according to the amount of curative substance
fed. In no case did the avocado oil or any of its irradiated
products fail to cause calcium anabolism. Chart XIV, page 57,
shows that the animals also gain in weight when placed on
the curative diet.
SUMMARY
0.4 cc. of unrefined pressed avocado oil is approximately
equivalent to 0.2 cc. of cod liver oil in its vitamin D
potency.
61
Irradiation of the avocado oil increased its vitamin
D potency approximately thirty-five per cent.
gO mg. of irradiated unsaponifiable matter is
approximately equivalent to 0.2 cc. of cod liver oil in its
vitamin D potency.
The percentage of unsaponifiable matter in avocado
oil was determined. Page 20
Phytosterol was prepared from avocado oil and
identified by qualitative tests. Page 24.
The phytosterol was irradiated and by means of test
experiments on rachitic rats, was shown to have an anti
rachitic function.
3 mg. of irradiated phytosterol acetate is approximately
equivalent to 0.2 cc. of cod liver oil.
The maximum amount of avocado oil that a rat can
tolerate per day was determined.
The special oils prepared by Mr. Sanborn (Table XII
and XIII, page 53 and 55) have not lost their anti-rachitic
function, but they have lost a considerable portion of their
growth promoting factor. (Chart XIV). Probably the vitamin
A has been destroyed in the purification of the avocado oil.
In conclusion, the investigation proved that avocado
oil and its irradiated products are decidedly anti-rachitic
in function.
BIBLIOGRAPHY
62
Bills, E. and McDonald, F, G. Journ of B. Chem. 68, 1926.
821 pp.
Hess, Alfred F. The Contribution of Biology. Chemistry, and
Physics to the Hewer Knowledge of Rickets. 1928.
1735 pp.
Hess and Weinstock. A Study of the Antirachitic Factor in
Human and in Cow1s Milk. 1927. 853 pp.
Hess, Weinstock, and Heilman. Antirachitic Value of
Irradiated Phytosterol and Cholesterol. 1925. 305 pp.
Hume,.Lucas, and Smith. On the Absorption of Vitamin D
from the Skin. 1927. 367 pp.
Jones, Murphy, and Helson. Vitamins in Clams. 1928.
652 pp.
Jones, Murphy, and Nelson. Vitamins in Oysters. 1928.
653 pp. ------------- -------
Kramer, Kramer, Shelling, and Shear. Cod Liver Oil Concentrate.
1927. 706 pp.
Leach. Food Inspection and Analysis. 1920. 524 pp.
Levinsohn. Rickets in the Negro. 1927. 961 pp.
McCollum and Simmonds. The Newer Knowledge of Nutrition.
1927. 430 pp.
McCollum, Simmonds, Shipley, and Park. A Delicate Test,
Biological, for Calcium Depositing Substances. 1927.
49 pp.
Outhouse, Macy, and Brekke. A Quantitative Comparison of
the. Antirachitic Factor in Human Milk and Cow1s Milk.
1928. 129 pp.
Price, Emma K. Vitamin D.
Ratliff, Mildred. Vitamin C.
Rosenheim and Webster. Rickets and Cholesterol. 1925.
1025 pp.
Schelly, Florence. Vitamin E.
65
BIBLIOGRAPHY (Cont»d)
Sherman and Smith. The Vitamins. 305 pp.
Sherman, Henry G. Chemistry of Food and Nutrition. 1927.
474 pp.
Steenboek and Black. Induction of Growth Promoting and
Calcifying Properties in Fats and their Unsaponifiable
Constituents by Exposure to Light. 1925. 1263 pp.
Waterman, Eugene. Vitamin B.
Watson, Ruth. Vitamin A.
Youzt, Ellsworth. Vitamin A.

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Asset Metadata

Creator Johnson, N. S (author)

Core Title Avocado oil as the source of vitamin D

Degree Master of Science

Degree Program Chemistry

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag agriculture, food science and technology,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor [illegible] (committee chair), Baldurn, M (committee member), Smith, Frank (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-785144

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