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alcohol. Alcohol is used here because water does not mix well
with the oil, but causes an emulsion formation, and the end point
is not clear. The acid number gives one an idea of the state of
freshness of the fat.

3. The saponification number or Koettstorfer number. The
saponification number is the number of milligrams of KOH
necessary to neutralize (to form a soap), with the fatty acids
derived from 1 gram of fat. Since fatty acids are monobasic one
molecule of potash neutralizes one molecule of acid, but each
molecule of fat required three molecules of KOH — since glycerine
esters or fats are tribasic.

The saponification value is determined by dissolving a weighed
amount of fat — about 2 grams — in a wide mouthed bottle
holding from 250 to 300 cc. Add 25 cc. of half normal alcoholic
KOH. Attach a reflux condenser and heat on a water bath for
30 minutes. Cool and titrate the excess of KOH with semi-
normal HCl, using phenolphthalein as the indicator. Sub-
tracting the acid necessary to neutralize, from 25 cc. gives the
saponification number.

Since fats are glycerine in combination with monobasic fatty



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152 CHEMICAL PHARMACOLOGY

acids, the saponification number will give indirectly the molecular
weight of the pure acid. This relationship is as follows:

Mol. weight Saponification number
Butyrin 302 557.3

Palmitin 806 208.8

Stearin 890 189.1

Olein 884 190.4

4. Unsaponifiable residue = Cholesterol and Phytosterol.
These previous numbers are of value in the calculation of the
molecular weight of acids only when we are dealing with pure
products. The numbers however are of value in determining the
nature of an oil, especially when taken in consideration with other
constants. One of these is the amount of unsaponifiable resi-
due. This residue consists mainly of cholesterols or phytosterols
which are soluble in petroleum ether, while glycerol, and potas-
sium hydroxide are not, and soap only slightly. Accordingly to
determine the unsaponifiable residue, after saponification cool
and filter oflf the soap — shake the solution with petroleum ether
in a separatory funnel, and evaporate in a desiccator to constant
weight, in a weighed dish. The residue represents the unsaponi-
fiable residue.

The following table gives the amount of unsaponifiable resi-
due in the more important fats.

Per cent, of
Unsaponifiable Matter

Lard 0.30 to 0.40

Castor oil 0.30 to 0.40

Human fat 0.33 to 0.00

Linseed oil 0.42 to 1 .00

Olive oil 0.46 to 1.00

Corn oil 1.35 to 2.90

Wheat fat 4.45 to 0.00

Shark oil 7.00 to 10.00

Sperm oil 37.00 to 41.00

Beeswax 52.00 to 56.00

The isolation and identification of the unsaponifiable residue,
is of importance in establishing whether or not a fat is of animal
or vegetable origin.



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IODINE NUMBERS 153

5. The iodine absorption number of fats (Hiibls number) . This
is the amoimt of iodine (per cent.) that a fat will absorb.
It is a measure of the unsaturated fatty acids in the fat. An
unsaturated (ethylenic) compound absorbs iodine after the man-
ner of ethylene :

C2H4 -j- I2 — ^02x1412

The resulting compound being^saturated.

To determine the iodine niunber the following solutions are
needed.

1 . 25 grams of pure iodine and 30 grams pure mercuric chloride,
in 500 cc. pure alcohol, free from unsaturated compounds.

' 2. A decinormal solution of sodium thiosulphate.

3. Potassium iodid 20 per cent, in water.

4. A 1 per cent, solution of starch paste as an indicator.
The determination is made as follows:

Weigh 0.3 gram of the fat in a glass stoppered bottle and dis-
solve in about 20 cc. chloroform and add 25 cc. of the iodine solu-
tion. Stopper the flask and set aside in the dark for 4 hours.
Wash into a flask for titration, with 10 cc. of the KI solution and
titrate with sodium thiosulphate solution. The diBference be-
tween the volume of thiosulphate needed and 25 cc. of iodine solu-
tion used will be the amount of iodine absorbed or the iodine
number.

The reactions involved are:

Each cc. N/10 thiosulphate represents 0.0127 gm. iodine.

I2 + 2(Na2S203 + 5H2O) = Na2S406 + 2NaI + IOH2O

The KI is added to prevent separation of the iodine in the
solid state when diluted with water. The mercuric chloride
forms:

Hg.Cla + I2 = Hg.ClI + ICl

The iodine chloride is perhaps the active agent in the addition,
and facilitates the process.
The iodine numbers of pure fats are :

Olein 86.2

Linolein 173.6

Linolenin 262.2



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164 CHEMICAL PHARMACOLOGY

Iodine Numbers of natural fats :

Linseed oil 175-205

Almond oil 145-150

Olive oil 80-88

Cottonseed oil 108-110

Codliveroil 107

Neat's foot oil 67-73

Palm oil 51

Cocoanut oil 8-9

Tallow... 35- 45

Lard 50- 70

Butter 26-38

Japan wax 4-10

Spermaceti 0.4

Unsaturation as evidenced by iodine absorption is a specific
instance or kind of unsatiu'ation and in no sense a general test
for unsaturation. The unsaturation in the case of fats and oils
is ethylenic — i.e. between carbon atoms. In aldehydes, ketones,

etc. which contain a carbonyl group yC = O, there is also

W
unsatiu'ation but iodine is not added to these. If hydrogen be
used, however, it reacts with the carbonyl as also with the
ethylenic linkage.

The reactivity in the one case and not in the other is due to
modification of the unsatiu'ated bonds by attached molecules
or atoms. This may be illustrated by the reactivity of the H
atom in water, alcohol and acid.

H.OH
CH3CH2OH
CH3COOH

The difference in reactivity in each case being due to the modi-
fying influence of the attached radical.

THE HYDROGEN NUMBER AND HYDROGENATED FATS

Under proper conditions hydrogen may be added to fats much
in the same way as bromine. This changes ill-smelling and



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ACETYL VALUE 155

tasting, cheap vegetable oils into more palatable products
resembling the more expensive animal fats. The process of hy-
drogenation is of great commercial importance. In some pro-
cesses finely divided metals such as nickel are used as catalyzer,
and some of the metal may remain in the finished vegetable lard.
Nickel may be absorbed from the gastro-intestinal tract; and it
is toxic, hence fats prepared in this way may be interesting from
a pharmacological point of view. The pure products are not
toxic, but if nickel remains in oil the latter may^ become toxic.
These hydrogenated fats are important economically.

THE REICHERT MEISSEL NUMBER

This represents the number of cubic centimeters of N/10 KOH
required to neutralize the volatile acids Uberated from 5 grams
of fat imder certain special conditions. The process of determin-
ing the amount consists in saponifying the fat with an alkali, then
adding an excess of a non-volatile mineral acid, distilUng and
titrating the volatile acids. Phenolphthalein is used as the indi-
cator. This method is especially useful in the examination of
butter fat for adulteration.

The Reichert Meissel numbers of the most important fats are:

Linseed oil 0.0

Goose fat 0.2

Tallow 0.5

Olive oil 0.6

Lard. . 0.7

Palm oil 5-7

Cocoanut oil 6-7

Croton oil 12-14

Butter fat ...... 25-30

No other fat contains as much volatile acid as butter.

THE ACETYL NUMBER

This is a measure of the number of hydroxyl groups in a fat.
The measurement of these depends upon the fact that substances
containing the alcoholic hydroxyl group react with the acetyl
group (CH3CO). The number of OH groups is arrived at by



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156 CHEMICAL PHARMACOLOGY

treating the fat with acetic anhydride and heat; when a reaction
takes place as follows:

^CHOH + 0<f -> ^HC.OOC.CHa+CHaCOOH

W ^CO - CHa W

The acetyl derivative of the fat is .stable in boiling water, and
by boiling in water, excess of acetyl anhydride is converted
into acetic acid. The acetylated fat can now be separated by
filtration and washed free from the acid, ^his acetylated fat
can be saponified according to the reaction:

ROCOCHs + KOH-^ROH + CH3COOK

In this way the amount of potash required for the saponifica-
tion can .be used as a measure of the acetyl groups, and hence of
the hydroxyl groups in the fat.

The number of milligrams of potash required t;o neutralize the
acetyl derivative of 1 gram of fat, is the acetyl value of that fat.

The following table gives the acetyl value of some common
pharmaceutic products:

Linseed oil . 0.4

OUveoil 10.5

Codliver oil 0.5

Spermaceti 4.5

Lard 2.6

Tallow (Beef) 2.5-9

Beeswax 15.0

Wool wax 0.23

Castor oil 0.15

The Elaidin Test for Fats (Gr. Elais— Olive Tree)

This test is distinctive for the oleic series. It depends on the
fact that oleic acid is changed from the cis to the trans form on
treatment with nitrous oxide, or liquid olein is converted into
solid elaidin — which is an isomeride of olein. Other acids of
this series are similarly transformed.

The Elaidin test is performed as follows:

(I) Place 10 cc. oil in a test tube and add 5 cc. nitric acid sp.
gr. 1.38-1.40 underneath it. Place a small piece of copper (0.2



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TESTS 157

gm.) in the acid. Leave at a temperature of not over 25°C. until
the following day, and observe frequently or

(II) 10 grams of oil are mixed with 5 cc. nitric acid sp. gr. 1.38.
and 1 gram of mercury, and the mixture shaken until the mercury
is dissolved. Set aside and shake again after about 20 minutes.
Note the time relquired f or solidification. This reaction is called
the "elaidic transformation."

Depending upon the amount of oleic acid present, the oils
vary in the length of time necessary for solidification.

Olive oil solidifies in about 60 minutes.
Peanut oil soUdifies in about 80 minutes.
Sesamer oil solidifies in about 185 minutes.
Riape oil solidifies in about 185 minutes.
Lard oil — inside two hours.
Linseed oil gives a red pasty froth.
Hempseed oil remains unchanged.

The temperature of the mixture should not exceed 25 degrees.
At best the reaction gives only an idea of the character of the oil.

The Bromine Test

This test depends on the fact that linolic, Unolenic and other
unsaturated drying and semi-drying oils form insoluble addition
compounds with bromine containing 6 or 8 atoms of this ele-
ment, which is insoluble in ether. Linolenic acid having three
double bonds yields a hexabrom derivative. The avidity of the
reaction can be measured also by the heat of bromination, which
runs parallel with the amount of bromine or iodine that a fat will
absorb. To determine the amount of bromine absorbed: 1 to
2 cc. of oil are dissolved in 40 cc. of ether and 2 cc. glacial acetic
acid. Cool to about 5°C. and add bromine drop by drop until
no more is absorbed.

The precipitate is collected on a weighed asbestos filter and
washed 4 or 5 times with ether, and dried in a steam oven. The
weight is directly proportional to the amount of unsaturated
acids in the fat.

Maumene or Sulphuric Acid Test

Fats of the Hnolic series on being mixed with sulphuric acid
evolve heat while those of the oleic series do not.



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158 CHEMICAL PHABMACOLOGY

The diflference in degrees centigrade between the initial tem-
perature and the temperature after the addition of sulphuric acid
under special conditions is known as the Maumen^ Number:
The test is carried out as follows:

Place a beaker of 150 cc. into a beaker of 800 cc. and pack the
space between with cotton. Weigh 50 grams of oil into the smaller
beaker. Place a thermometer in the oil and run in 10 cc. con-
centrated H2SO4 from a burette at the same temperature as the
oil. Stir the oil with the thermometer while the acid is running
in. The temperature rises quickly, and remains at the high point
a sufficient time to permit observation. The maximum point
should be noted.. The initial temperature subtracted from the
maximum gives the Maiunen^ number.

RANCIDITY OF FATS

Most fats but especially those containing unsaturated adds
on exposure to the air become rancid and develop a disagreeable
smell and taste. The unsaturated fatty acids are converted into
others containing a smaller number of carbon atoms. Among
the decomposition products aldehydes, alcohols, hydroxy acids
and esters have been found. The actual cause of rancidity is
but Httle understood. Oxygen, Ught, and heat, and moisture,
facihtate the process which is probably initiated by enzymes and
bacteria, while free acid is Uberated in the process.

Acids may be developed without rancidity as is often seen
in cocoa butter which is frequently acid but rarely rancid.

THE SIGNIFICANCE, USES AND FATE OF FATS

Fat is found in varying amounts in all forms of living matter.
This may not be seen in microscopic sections or when stained with
Sudan III, osmic acid and other*fat stains but organic substances
when extracted with ether and other fat solvents, always yield
a lipoid residue on evaporation. After anesthesia for an hour
with chloroform, sudan III shows that fat droplets are distinctly
present in the cell, while chemical analysis shows that there is
no greater amount than before the anesthesia. It is differently
distributed after the anesthetic.

In the economy of both plants and animals, fats are connected



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SIGNIFICANCE OF FATS 159

with nutrition. They are readily stored and provide a food
reserve which in animals is used in cases of food deficiency.

They act as protectors to the proteins of the body, sparing the
protein from oxidation. They also act as lubricants to the skin
and aid in keeping it soft and pliable. If the lipoid material is too
frequently and too vigorously removed from the skin, as is some-
times done by the excessive use of highly alkaline soaps, the skin
becomes dry and eczematous. In such cases the judicious
use of oils externally is very beneficial. Many fats are used in
emulsions for this purpose. Some fats because they are decom-
posed into slightly irritating lAaterials in the intestines are used
as cathartics.

In the protoplasm fats are distributed very finely as in milk.
None of the ordinary fat tests will detect fat when it is so finely
divided and protected. The fat in the cells in this condition may
also act as a protective to the essential part of the cell. In phos-
phorus poisoning and in other conditions classed as fatty de-
generations, the fat is run together and so loses its protective
properties. In these conditions there is no increase in the actual
body fat, but simply a redistribution of it. Why one person is
fleshy — or the body retains a considerable amount of fat — while
another, is lean cannot be explained further than that the funda-
mental properties of the protoplasm is different. This may de-
pend on the physiological activity of some endocrine gland either
acting on the seats of oxidation directly or through the nerves.
It is known that basal metaboUsm is distinctly higher in hyper-
thyroidism, and lower in hypothyroidism, and in other conditions.

Oxidation furnishes the heat necessary for the body and fats
are the heat producing foods par excellence, one gram of fat pro-
duces 9.3 calories. Fats also act as a mantle and since they are
poor conductors they aid in heat conservation by preventing
evaporation and radiation. In cases of obesity this property
may be a hindrance rather than a benefit. Fats also act as pack-
ing material for such organs as the kidney, which is partially
embedded and held in place by a cushion of fat.

In plants fats are foimd in greatest amounts in the seeds and
propagative organs. Their function here is protective, to pre-
vent desiccation which would prevent germination, they also
serve as nutritive material. Seeds contain lipases which may



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160 CHEMICAL PHARMACOLOGY

either hydrolyse the fat into fatty acids and glycerine or syn-
thesize the fats from the same materials.

Regarding the origin of fat in the plant little is definitely known.
In many cases there seems to be strong evidence that it originates
from tl;ie carbohydrates. Certain seeds like the almond, and
castor bean, and olive in the green state are rich in carbohydrates
and poor in fats, but as they ripen the carbohydrate decreases
and the fat increases. Glucose, sucrose, mannite, starch and
other carbohydrates, have been observed to change in this way.
Ivanow, in case of flaxseed represents the changes taking place
as follows:



yGlycerine v

Carbohydrate ^ yFat.

^Satiu*ated Unsaturated^

fatty acid fatty acid

The reverse change is supposed to take place diu*ing germination.
Miller found in case of the sunflower that the cotyledons in the
resting state contained 1 per cent, free fatty acid while in the
seedling there was 30 per cent, fatty acid. These fatty acids
disappear, that is, are used by the plant in the following sequence;,
linoleic, linolic, oleic and finally palmitic; that is the more im-
saturated acids are used first. There is some difference of opin-
ion as to the changes in the original fat during germination, but
one acid may be transformed into another.

It has been suggested that starch may arise from oleic acid as
follows:

C18H34O2 + 270 = 2(C6Hio06) + 6CO2 + 7H2O

Fats may also arise from protein, but the proof of this is not
so definite in the plant as in the animal. Fats may also be trans-
ported in the plant from one region to another, similar to fatty
infiltration in the animal.

ORIGIN OF FAT IN THE ANIMAL

1. It may arise from the fat of the food. Proof of this is found
in the fact that when linseed oil, rape oil, mutton fat and the
like are fed to dogs — these fats can be recognized in the fatty
deposits of the tissues of the animal. Experiments have shown



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ORIGIN OF FAT 161

that the fat of dogs fed on Unseed oil, melts at 0° — while those
fed on suet was solid at 50°C.

2. From carbohydrates; animals have been fed on a carbo-
hydrate diet, and the carbon retention has been shown to be in
the form of fat. For example: Rubner fed a dog weighing 5.89
kg. on starch, sugar, and fat that had a total carbon content of
176.6 grams. During the period the animal excreted 87.1 grams
of carbon, there was thus a retention of 89.5 grams. The fat
of this diet had a carbon content of 3.6 grams. The animal ex-
creted 2.55 grams nitrogen = 16 grams protein — (2.55 X 6.25).
On the improbable assumption that all the carbon of this ex-
creted protein was retained in the body, this would be 8.32 grams
C (16 X 0.52) (52 per cent. C in proteins) so that 8.32 + 3.6 =
12 grams, could originate from other sources than carbohydrate
leaving 89.5 — 12 = 77.5 grams of carbon that could arise only
from the carbohydrate and could be retained only as carbohy-
drate or fat. The greatest possible amount of glycogen that
could be stored from this would be 78 grams or 34.6 C so that there
would still remain 42.9 grams of C that could be stored only as
fat. This calculation is based on the fact that glycogen
is stored equaHy between the liver and the muscles. The liver
rarely exceeds 4 per cent, of the body weight and only in excep-
tional cases will the liver glycogen = 17 per cent, of the weight
of the organ.

Numerous other fattening experiments have convinced physi-
ologists that fats can be formed in the animal body from carbo-
hydrate. The chemistry of this change is not understood, and
cannot be imitated in the laboratory. See Lusk, Science of
Nutrition, 3d Edition. The following hypotheses have been
proposed in that the process starts with pyruvic acid. Lactic
acid arises from the sugar and may be converted into pyruvic
acid by oxidation. The pyruvic acid unites with an aldehyde to
form higher fatty acids:

I. R CHO + CH3CO COOH = R CHOH CH2 CO COOH.

II. R CHOH CH2 CO COOH + = R CHOH CH2 COOH +

COa and

III. R CH2 CH2 COOH.

may also be formed on further oxidation.
11



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162 CHEBaCAL PHABMACOLOGT

This gives some idea of how higher fatty acids may be formed
in the plants. The glycerol necessary to form fat from the fatty
acid may be synthesized in the plant in a manner unknown to
the chemist. That it may be formed from the elements has been
shown by Priedel and Silva through the following steps:

CH3COOH -^CHaCO.CH,-^

Acetic acid Acetone

CH,.CH0H.CH3 -^CH,.CH:CH2
Propyl alcohol Propylene

CH,.CHCl.CH,Cl-^CH2a.CHCLCH,Cl-^
Propylene chloride Trichlorhy drin
CH2OH.CHOH.CH2OH
Glycerol

FATS FROM PROTEINS

It has been shown quite definitely in feeding experiments that
fat may be formed from protein. There has been considerable
difference of opinion on this question. Pettenkoffer and Voit
claiming a distinct formation while Rubner questioned the com-
putation on the basis that they had used the ration of carbon to
nitrogen in protein as 3.68 instead of 3.28 which he believed to
be the correct figure. Cremer, however, showed by experiment
that fat may be formed from protein and his results have been
amply confirmed. His experiment is as follows:

A cat was starved for a number of days. It was then fed 450
grams of meat a day. The animal was kept in a respiration
chamber and the CO2 in respiration measured and the excreta
analysed. There was a daily excretion of 13.0 grams nitrogen —
41.6 grams of protein carbon (13 X 3.18). However only 34.3
grams of carbon was eliminated. 7.3 grams or 17.5 per cent, of
the carbon taken in was retained. In 8 days 58 grams of carbon
was retained. If this were stored as glycogen it would make 130
grams, but in the total animal at this time there was found only
35 grams of glycogen. The balance must have been stored as fat.

This subject has also been investigated by Atkinson and Lusk
who have shown by calculations based on respiratory quotients
and heat production as measured by the respiration calorimeter
that fat is produced from protein in the dog after the ingestion
of large quantities of protein.



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ORIGIN OF FATS 163

THE NEED OF FATS IN GROWTH

The normal growth of an animal depends upon something in
addition to the requisite number of calories of fats, proteins and
carbohydrates. The fat must be of a certain source and contain a
growth promoting substance "A," or what has been called vita-
mine. All fats do not contain this vitamine. It is especially
abimdant in butter fat, beef fat, egg yolk, and cod liver oil.
Animals fed on a diet in which oHve oil or almond oil suppUes the
fat, do not grow, and soon will die if such diet is continued.
However, even when death is near, the substitution of vitamine
containing fat, immediately restores normal health and growth.
The nature of this substance is not known. The term vitamine,
suggests that they are amines, but such is not the case. The term
vita, McCoUum thinks, gives an importance to these essentials,
greater than other equally indispensable constituents of the
diet. He suggests until mor^ definite knowledge is obtained, the
term fat soluble ''A" be applied to the vitamine essential growth
promoting ingredient of fats, and to other Uke substances which
are soluble in water, water soluble " B. ''

THE FATE OF FATS IN THE BODY

Fats are easily and completely oxidized in the body and are a
great source of body heat. They are absorbed after saponifica-
tion and resynthesized again in the body, probably by an enzyme.
In the dog 10-20 per cent, of the fat of a meal is absorbed in four
hours, about 30 per cent, in seven hours and 86 per cent, in 18
hours. After excision of the pancreas, or disease of it, fat ab-
sorption is markedly retarded but not aboUshed.

In man the feces contains 0.5 to 1.5 grams of fat in starvation,
while on ordinary diet containing about 120 grams fat, 3 to 7
grams is excreted.

Normal urine contains no fat, but in diseased conditions
variable amounts may be found. The condition is known as
lipuria and may occur after excessive eating of fat, after cod
liver oil, in fat emboUsm occurring after fractures, in phosphorus
poisoning and other fatty degenerative processes, in prolonged
suppuration, chronic Bright's disease, diabetes, chronic alco-
holism, in wasting diseases, diseases of the pancreas, obesity,
leukemia, and in mental diseases.



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164



CHEMICAL PHARMACOLOGY



XVin. WAXES

The waxes are esters of higher monatomic alcohols or sterols
such as cetyl alcohol, CieHssOH, myristic alcohol, CsoHeiOH,
or cholesterol C27H46OH, and one of the higher fatty acids.
Spermaceti is a wax, obtained from a cavity in the head of the
sperm whale, and consists mainly of cetyl alcohol and palmitic



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