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A Study of Alcohol Tolerance in
Animals and Humans

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A STUDY OF ALCOHOL TOLERANCE
IN ANIMALS AND HUMANS

by

EDWARD STEPEEN CES‘I‘ARIC

A THESIS
Submitted to the School of Graduate Studies of Michigan
State College of.Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE

Department of Chemistry

1951

(LEV-11;". 1* W1 r

Twit)";
£411

“I ‘/ "7 f, 5'}

:m

CONTENTS
Page
INTRODUCTION . . . . . . . . . . . . . . . . . . l
HISTORICAL . . . . . . . . . . . . . . . . . . . 4
IEXPERIEENTAL . . . . . . . . . . . . . . . . . . 6
Determination of Alcohol in Water-Alcohol
and Blood—Alcohol Solutions of Known

Concentrations . . . . . . . . . . . . . . 7

Experimental Work on Rate . . . . . . . . lO

‘8

Experimental Work on Human Subjects . . .
SUMMARX . . . . . . . . . . . . . . . . . . . . . 22
DISCUSSION . . . . . . . . . . . . . . . . . . . 25
BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . 28

LIST OF TABLES

Table I
Determinations of Known Water—Alcohol
Solutions . . . . . . . . . . . . . .

Table II
Determinations of Known Blood—Alcohol
Solutions 0 O O O O O O O O 0 O O O 0

Table III
Liquid Consumption . . . . . . . . .

Table IV
Oxygen Uptake . . . . . . . . . .

Table V
Alcohol Concentration of Tissues . .

Table VI
Alcohol Concentration of Tissues . .

Table VII
Quick's Test-Human Subjects . . . . .

Table VIII
Comparison Ratios . . . . . . . . . .

Table IX
Comparison Ratios . . . . . . . . . .

Page

10

12

15

17

19

21

24

25

ACKNOWLEDGEIENTS

The author wishes to express his sincere gratitude
to the following gentlemen:

Dru C. A. Hoppert, Professor of Chemistry; Dr. C. W.
Muehlberger, Director of Crime Detection Laboratory
of Michigan.Department of Health and Mr. R. F. Turner,
Associate Professor of Police Administration for their
invaluable assistance and consultation.

Dru W. D. Collings, Associate Professor of Physiology
and Pharmacology and Dr. R. A. Fennell, Professor of
Zoology for their many helpful suggestions.

Dr. R. U. Byerrum, Assistant Professor of Chemistry,
for his invaluable assistance and many helpful suggestions.

To the twenty—four subjects who gave their time and
cooperation to make this study possible.

And also to the National Safety Council, Committee on
Chemical Tests for Intoxication, Chicago, Illinois
whose financial aid towards this fellowship made this
study possible.

INTRODUCTION

1 by the liver has been studied

The oxidation of alcohol
in many ways by a number of persons. In 1937, Lundsgaard (1)
showed that alcohol disappeared very rapidly from the isolated
liver, whereas with perfused hind limb muscle no oxidation of
alcohol could be demonstrated. In 1937, Dontcheff (2) showed
that the oxidation in the fasting white rat Was dependent up—
on the stage of metabolism through which the rat was going.
During the first stage, when primarily carbohydrate was being
metabolized, the rate of oxidation of the alcohol was quite
high. In the next stage, during which a major portion of the
fat was being utilized, the rate was comparatively low. In
the final stage, in which primarily protein was being metabo—
lized, alcohol was again found to be oxidized at a rapid rate.
In 1956, Rosovskaya (5) demonstrated that hyperthyroidism
caused little acceleration of alcohol oxidation, and in 1959,
Mirsky and Nelson (4) showed that it was the liver and not
the pancreas which influenced the oxidation of alcohol. It
was substantiated that the rate of alcohol utilization was
apparent. In the eviscerated rabbit it was shown that alcohol
was not utilized even if glucose or insulin was added, which

indicated that muscle did not utilize the alcohol.

1
The alcohol referred to throughout this work is ethanol
unless otherwise specified.

LeBreton (5) showed that when fasting dogs were given
small doses of alcohol, about ninety per cent of the oxygen
consumed Was accounted for by the oxidation of the alcohol.

Alcohol tolerance, as viewed by Newman and Lehman (6) and
Newman and Card (7) is primarily a tissue tolerance whereby
the cells of the central nervous system acquire the ability to
function effectively in the presence of alcohol. Later in
1941, Newman (8) further demonstrated this by habituating dogs
to alcohol. He then gave test doses of alcohol to habituated
as well as to non—habituated dogs. He found that the former
showed a markedly greater ability to control their neuro—muscu—
lar apptitudes. The habituated animals "sobered up" more rapid—
'ly despite the fact that their blood alcohol levels fell at the
same rate as that of the controls.

Richter (9) showed that rats preferred alcohol concen—
trations of one to six per cent in water; but when forced to
take larger amounts of alcohol, they reduced their food con—
sumption almost directly proportional to the caloric intake
obtained from the alcohol. Since the rats grew and thrived
under these circumstances, the results indicated that the al—
cohol may have replaced large amounts of food. Mitchell and
Curzon (10) showed that the alcohol does possess a distinct
energy food value. The energy of the alcohol is more sparing—
ly utilized when carbohydrate food is simultaneously ingested.

They postulated the oxidation of both alcohol and carbohydrate

is promoted by thiamine, which is essential for the metabolism
of carbohydrate.

Possibly because of the fact that alcohol supplements the
diet for certain necessary foods, the liver becomes damaged in
excessive drinking. According to Iziri (11) alcohol has a
specific toxicity for the liver. Later Gates (12) working
with human subjects using the bromosulfalein test, showed that
alcoholics, after prolonged and continuous drinking had liver
impairment. Chaikoff, et al (13) working with dogs showed that
severe fatty degeneration and even cirrhosis appeared in the
liver where high protein diet with alcohol was given, and Lowry,
et a1 (14) working with rats showed that with a low protein
diet the substitution of twenty per cent alcohol for drinking
water produced cirrhosis.

The foregoing experiments may lead one to suspect that
the alcohol, itself, is the cause for the liver disorder. How—
ever, as pointed out by Jolliffe (15), Jolliffe and Jellinek
(16), and Remington and.Leitner (17) alcohol "per se" is not
the cause. Inebriety and cirrhosis are definitely associated;
but a combination of circumstances (8. g. alcoholism plus cer—
tain nutritional deficiencies)is required to produce liver

damage.

HISTORICAL

Scientists and law enforcement agencies have made con—
siderable progress in the development of methods and special
apparatus for the detection of intoxication. That this is a
fact, may be concluded from a statement from the F.B.I. Law
Enforcement Bulletin of April 1, 1958, p. 15, "Scientific in—
vestigators have pointed out and repeatedly verified the fact
that the concentration of alcohol in the body fluids is one of
the most reliable and objective criteria of intoxication.

Many of these investigators have determined the relationship
between brain alcohol concentrations and concentrations in the
spinal fluid, urine, saliva, and breath, so that the degree of
alcoholic influence can be evaluated closely when the concen—
tration of alcohol in the breath or in the body fluids is
known.“ However, the methods for the detection of intoxication
(Drunkometer, Intoximeter, and the Alcometer)(27) still provide
no satisfactory explanation as to why some individuals are
capable of consuming more alcohol than others before reaching
the same degree of intoxication. The phenomenon of “tolerance"
is quite complicated. Both the psychological factor involving
the state of mind in which he may be and the physiological
factor involving the type and amount of food consumed prior to

alcohol intake may play an important role in so—called "tolerance."

Even with these complicating factors, certain individuals
must consume more alcohol than others in order to reach the
same degree of intoxication or blood alcohol levels. This is

evident even under comparable conditions of alcohol consumption.

EXPERIMENTAL

Investigations by'Dontcheff (2), Rosovskaya (S), and
Mirsky and Nelson (4) showed rather conclusively that the
liver is an important organ in the metabolism of alcohol.

For these reasons in the present investigation it was decided
to study the liver to determine whether there is any cor;
relation between its function and-tolerance of an individual
towards alcohol.

In conjunction with this the alcohol concentrations of
the blood, liver, and kidney were determined in order to as—
certain any significant difference in the ratios of alcoholic
concentrations of these tissues for the tolerant and non—
tolerant animals.

In order to make a preliminary investigation by which
the liver promotes "tolerance" in certain individuals and not
in others, the latter part of this experiment was carried out
using Quick's hippuric acid function method. It was hoped
that a correlation might be found between the capacity of the
liver to form hippuric acid and the capacity to metabolize
alcohol. In this part of the experiment, "tolerant" and "non~
tolerant" human subjects were chosen from individuals who had
participated in previous experiments. (24) The amount of hip—
puric acid formed by the subjects before and after the ad—

ministration of alcohol was used as the basis upon which the

human liver function was determined.

_ a l

To be familiarized with the procedure for the determin-
ation of alcohol concentrations of solutions by the modified
Nicloux method, (18) a number of trial determinations were
made. The modified Nicloux method involves the distillation
of the alcohol from a Kjeldahl flask through a steam trap in—
to a Liebig condenser. The distillate is collected in a test
tube containing an acid—dichromate2 mixture, boiled for fifteen
minutes to ensure complete oxidation, and subsequently titrated
with sodium thiosulfate using one per cent starch solution as

an indicator. The reactions involved may be represented as

follows:
1 2K Cr 0 8H so so H OH wees coon 2Cr so
) 2.27" 24" 25 s ’l 2-( 435"
2 1.
K2504 I 1 s20 .
2 K 6 s
) 2Cr207 ,1 KI ,1 mzsoé ..... 512 .4 4K2 o4 ,1 Cr2(SO4)3 ,1 7H20
s 5 SN r
) 12 I 9.23205 -.— 6Na28406 ,z mar

Determination of Alcohol in WaterfAlcohol and Blggd:Alpghpl
Solutions of Known Concentrations

 

 

A number of known water-alcohol solutions were prepared
for the preliminary determinations. This was done first by
redistilling alcohol four times and determining the final
concentration of the solution by the use of a calibrated

specific gravity_bottle§, and comparing to water at 200/2000.

 

2The acid—dichromate mixture consisted of five milliliters of
concentrated sulfuric acid and five milliliters of the 0.4S4N
potassium dichromate.

5The specific gravity bottle and all other equipment used were

calibrated before used.

The specific gravity was then converted to per cent ethanol
and found to be 93.46 per cent by weight. The alcohol was
sealed in two ounce bottles until ready for use in preparing
the known solutions. From this stock solution the known water—
alcohol solutions were prepared and their concentrations deter-
mined. The exact composition of the solutions was determined
bu the difference in the weight of the pipette before and
after delivery:

Wt. of the alcohol delivered x 93.46% _ of alcohol b wt w w
Total wt. of solution ‘ % y ' /

 

Errors in the determination may result from a number of
sources as indicated by Bennett (24).

The strength of the potassium dichromate solution was
0.434 N (18). Five milliliters of this solution was equivalent
to twenty-five milligrams of alcohol if completely reduced.
The sodium thiosulfate used was approximately 0.05 N. A blank
titration was made to determine the exact amount of sodium
thiosulfate equivalent to five milliliters of the 0.434 N
potassium dichromate. The amount of alcohol oxidized was then
calculated as follows:
A = ml. of Nazszo:5 used in sample titration
B

ml. of Na25205 used in blank

B - A

 

x 25 mg. = mg. of alcohol in sample

Grade 0f al°0h01 X 100 I per cent alcohol by Wt.
wt. of sample

 

-8-

Table I gives the results of the percentages of recovery.

TABLE I

DETERMINATIONS OF KNOWN WATEfieALCOHOL SOLUTIONS

Water samples of known alcohol concentration by weight
were prepared by the method described in the experi—
mental part of this work. The determinations were then
run on these samples using the modified Nicloux method.

 

 

Sample %—A1cohol %~A1cohol in Per cent
No. in known determination Recovery
1 0.219 0.216 99.01
2 0.219 0.214 97.94
3 0.219 0.216 99.01
4 0.219 0.204 93.04
5 0.219 0.212 96.78
6 0.219 0.210 96.11
7 0.219 0.210 96.11
1 0.0685 0.0685* 99.99
2 0.0685 0.0675 98.58
3 0.0685 0.0674 98.37
4 0.0685 0.0680 99.33
5 0.0685 0.0675 98.51
6 0.0685 0.0677 98.88
1 0.124 0.121 97.61
2 0.124 0.121 97.61
3 0.124 0.120 96.68
4 0.124 0.122 98.14
5 0.124 0.110 89.05

 

‘I

This determination did not reach exactly 100 per cent

After the Water alcohol solutions were analyzed, a number of
blood samples of known alcohol content were used to secure the
necessary accuracy in preparation for the analysis of tissue

samples of the rats. These results are tabulated in Table II.

TABLE II

DETERMINATIONS OF KNOWN BLOOD-ALCOHOL SOLUTIONS

Blood samples of known alcohol concentration by weight were
prepared by the method described in the experimental part
of this work. The determinations were then run on these
samples using the modified Nicloux method.

 

 

Sample %_Alcohol %flAlcohol in Per cent
No. in known determination Recoveryi
1 0.0882 0.0854 96.73
2 0.0882 0.0854 96.62
3 0.0882 0.0852 96.51
1 0.0566 0.0556 98.11
2 0.0566 0.0556 98.11
3 0.0566 0.0552 97.49
1 0.0310 0.0294 94.68
2 0.0310 0.0299 96.45
3 0.0310 0.0297 95.81

 

Experimental Work on Rate

In this study the determination of the "tolerance" was
made with twenty-four rats. The twenty—four rats were divided

into three groups (10—10—4), all fed the same stock ration,

-10....

which contained the following ingredients:

yellow corn meal 32.50% by Wt.
— ground wheat 25.00% by wt.
white milk powder 22.50% by wt.
linseed oil meal 10.00% by wt.
alfalfa 6.00% by wt.
Brewer's yeast 3.00% by wt.
table salt 1.00% by wt.

Five per cent alcohol was fed to one group of ten; ten per
cent alcohol Was fed to the other group of ten, and the re—
maining four were used as controls being fed plain water.
Each group consisted of an equal number of males and females.
Accurate records of food and liquid consumption were kept on
twelve of the rats (six males and six females, four from each
group) for a period of 128 days (19). During the first week
of feeding a definite loss of coordination and lower food
consumption for those receiving alcohol were noted. However,
as"tolerance" was gradually developed, their coordination
returned to normal, and food and liquid consumption increased.
Table III gives a tabulation of the average amounts of food

and liquid consumed per one hundred grams of body weight.

_ 11 _

TABLE III
LIQUID CONSUMPTION

The figures are given in milliliters of liquid consumed per

100 gram body weight per day per rat. The percentages shown
in parenthesis were calculated by using the control animals

as 100%

 

 

 

 

 

 

Female Rats Male Rats
Control 5%' 10% Control 55 10%
13.26 17.35 12.31 5.97 11.68 7.95
9.01 16.46 14.70 5.83 14.75 8.91
Av. 11.135 16.91 13.51 5.90 13.21 8.43
(150%) (121.2%) (225.5%) (142.9%)

 

FCCD CONSUNPTION

The figures are given in milligrams of food consumed per 100
gram body weight per day per rat. The percentages given in
parenthesis were calculated using the control as 100%

 

 

 

 

 

Female Rats Male Rats
Control 5% 10% Control 5% 10%
6.57 4.48 4.74 4.43 5.69 4.85
6.53 5.71 5.19 ggiz 6.87 4.59
AV.6.55 5.595 4.96 4.80 6.28 4.62
(85.4%) (75.5%) (130.8%) (95.2%)

 

-12...

After the required period of time for the development
of tolerance elapsed, the rats were sacrificed. Three hours
prior to sacrificing, each rat was given three grams of a1—
cohol per kilogram of body weight by means of a stomach tube
(20). (The alcohol was administered in 33—1/3% concentration.)
At the end of the three hour period, the alcohol was assumed
to be in a state of equilibrium throughout the body (20).

The metabolic rate of oxygen consumption of the liver Was
6 determined by the manometric Warburg technique. Before the
animals were sacrificed the Barcroft~Warhurg vessels were part~
ially prepared by pipetting two milliliters of Krebs—Ringer
Phosphate solution (pH 7.4) into the larger portion of the
vessel4 (21,22,23). The vessels were then set aside until the
tissue slices were prepared. This procedure was essentially
as follows: Immediately after the animal was sacrificed, the
blood was withdrawn and placed in beakers containing sodium
fluoride and sodium oxalate (a preservative and an anti—
coagulant) and refrigerated. The sodium fluoride and sodium
oxalate Was a one to one mixture, approximately one milligram
of the mixture being used for every milliliter of blood. The

mixture was wetted down with a few drops of distilled water

 

and spread along the sides of the beaker and set to dry. The

4
The potassium hydroxide was not put in at this time to
eliminate any possibility of contamination of the tissue
by placing it in contact with the potassium hydroxide.

-13...

kidney and liver were then removed and frozen until further
use. For the Warburg determination the middle lobe of the
liver was used.

The tissue slices were prepared by cutting cross—section
areas of the liver approximately one centimeter in diameter
and placing them between two microsc0pe slides which were
frosted by rubbing with emery cloth. A razor blade was then
passed between the top slide and the tissue to obtain slices
of about 0.2 millimeters thickness. The slices were weighed
accurately to about 100 milligrams per vessel. The weighing
Was done as quickly as possible without suspending the slices
in any liquid to prevent diffusion of the alcohol and were
transferred to the Warburg vessel. The elapse of time between
slicing of the tissue and placing them in the Warburg vessel
was approximately ten to fifteen minutes. Two—tenths milli—
liters of 30% KOH was pipetted into the center Well and small
strips of filter paper were placed into the well to increase
the absorption area. They were then attached to the manometers
and gassed with oxygen. After the gassing was completed, the
manometers were placed on a constant water bath for ten minutes
to attain constant temperature throughout the system. When
constant temperature was attained the stopcock was closed and
the shaking begun. The shaking was continued for one-half hour
with readings being taken at ten minute intervals. A thermo-
barometer was prepared in exactly the same manner except that

the tissue slices were omitted. ‘Dry weights of the liver were

-14-

obtained by weighing accurately 300—500 milligrams of the
tissue slices and drying them to constant weight between 1050
and 1100 overnight.

It was found that rats which received the 10% alcohol
solution had the lowest oxygen uptake, whereas those receiv—

ing water had the highest. The results are shown in Table IV.

TABLE IV

OXYGEN UPTAKE (Q02)

Q0 is expressed as oxygen uptake per milligram of tissue
peg half hour. The percentages given in parenthesis were
calculated using the control as 100% uptake.

 

Easels
Control 5% ' 10%
(2 animals) (3 animals) (3 animals)
~2.70 t 0.150 -l.89 : 0.895 -1.64 t 0.590
(100%) (70.0% (60.7%)
Iz'Tale
Control 5% 10%
(2 animals) (4 animals) (4 animals)
“2018 i 00152 ”1.96 t- 09524 —1060 i 0.285
(100%) (89.9%) (75.4%

- 15 a

The alcohol concentration of the liver, kidney, and
blOOd was also determined by the modified Nicloux method
(18). In the preparation of the tissue for the determin—
ation/a known weight of the tissue was sliced into small
pieces. These were then transferred quantitatively to a
Waring Blender and macerated with a minimum amount of water.
When the maceration was complete, the tissue homogenate was
again quantitatively transferred to twenty—five milliliter
volumetric flasks and brought to volume with distilled
water. The foregoing was the method used for the liver and
kidney. However, for the blood, a measured volume was trans-
ferred to ten milliliter volumetric flasks and brought to
volume with distilled water. The average amount of blood ex—
tracted from the rats was about seven milliliters. Aliquots
of the prepared tissues were then used for the determination.
Because of the small quantity of sample available, it was
necessary to change the concentrations of the reagents. In~
stead of using five milliliter samples, three milliliter
samples were used and the strength of the potassium dichromate
was reduced to 5/5 (or 0.261 N); likewise the sodium thio~
sulfate was reduced to approximately 0.03 N. Therefore five
milliliters of the solution was equivalent to fifteen milli—
grams of the alcohol. The calculations were carried out in

the usual manner —

_ 16 _

 

fie— x 15 = mgs. of ethyl alcohol

Grams of alcohol x 100 _
Wt. of sample ’

 

per cent of alcohol

Table V gives the results of the alcohol concentrations of
the tissues of individual rats and Table VI the average of
each group.

TABLE V

ALCOHOL CONCENTRATION OF TISSUES

The concentrations are given in percentage of alcohol by
weight in the tissues.

 

 

 

No. Blood Liver Kidney

52 ANIMALS

(Female)
1 0.0724 0.0718 0.0288 0.0226 0.146 0.134
3 0.0025 0.0028 0.113 0.108 0.159 0.169
1a. 0.0772 0.0791 0.192 0.194 0.133 0.142
2a. 0.148 0.143 0.124 0.108
3a. 0.0279 0.0269 0.051 0.0556 0.0495 0.0498

(Male)

2. 0.0264; 0.0262 0.1057 0.113 0.148 0.154
4. 0.0115 0.0159 0.0399 0.0416 0.114 0.111
7a. 0.110 0.109 0.114 0.109 0.122 0.116
Ba. 0.109 0.0989 0.0546 0.0608 0.0911 0.0833
9a. 0.0255 0.0361 0.114 0.110 0.124 0.136

_ 17 _

TABLE V (Continued)

 

 

 

 

N0. Blood Liver Kidney
10g ANIMALS

(Female)
5. 0.0706 0.0706 0.0458 0.0414 0.159 0.149
7. 0.0950 0.0959 0.1700 0.158 0.285 0.285
48. 0.0885 0.0789 0.162 0.170 0.555 0.540
58. 0.0149 0.0145 0.0688 0.0660 0.104 0.099
6a. 0.0179 0.0174 0.0262 0.0209 0.0951 0.100

(Male)
6. 0.0202 0.0192 0.100 0.101 0.0898 0.0864
8. 0.0187 0.0196 0.0984 0.104 0.0694 0.0743
10a. 0.167 0.168 0.135 0.147 .
11a. 0.0257 0.0240 0.0184 0.0224 0.0875 0.0927
128. 0.0701 0.0705 0.148 ' 0.150 0.154 0.148
CONTROL ANIMALS

(FPMale)
9. 0.0975 0.0960 0.105 0.106 0.151 0.148
11. 0.0875 0.0884 0.0225 0.265 0.521 0.510

(Male)

10. 0.0279 0.0284 0.0790 0.0808 0.0927 0.0859
12. 0.0718 0.0718 0.0587 0.0287 0.0555

0.0529

 

_ 18 _

The data in this table was obtained by determining the

TABLE VI

ALCOHOL CONCENTRATION OF TISSUES

average alcohol concentration for the males and females
from Table V, then determining an over all average of

each group.

The concentrations are given as percentages

 

 

 

 

 

 

 

 

 

 

 

by weight.
Blood Liver Kidney
CONTROL ANIMALS
Female 0.0925 t 0.01 0.1755 1 0.11 0.2258 1 0.14
Male 0.0500 t 0.02 0.0579 t 0.04 0.0502 s 0.05
Average 0.0712 130.02 0.1157 s 0.08 0.1450 1 0.10
5% ANIMALS
Female 0.0455 t 0.07 0.1055 1 0.15 0.1215 t 0.09
Male 0.0559 t 0.12 0.0862 t 0.09 0.1200 1 0.05
Average 0.0505‘I”0.10 0.0959‘i 0.15 0.1208 2 0.07
10% ANIMALS
Female 0.0555 s 0.09 0.0929 t 0.17 0.1954 1 0.28
Male 0.0501 s 0.14 0.1025 2 0.11 0.0755-t 0.17
.Average 0.0582 2 0.12 0.0977 i 0.14 0.I344 i 0.26

 

-19..

Experimental Work on Human Subjects

The third part of the experiment was done with human
subjects. The purpose was to ascertain whether there was
any correlation between the formation of hippuric acid and
the degree of "tolerance“ after the administration of al—
cohol. From previous tests (25) a number of individuals
who were known to have a high "tolerance" and those of a
low “tolerance" were selected. Quick's liver function test
(24,26), for the formation of hippuric acid, was used as
the basic criterion. This test is based upon the synthesis
of hippuric acid by the liver from benzoic acid and amino—
acetic acid. The hippuric acid is excreted in the urine
normally at a nearly constant rate. First a normal liver
function test was carried out with each subject. Then after
several days had elapsed the subject was allowed to-ingest a
certain amount of alcohol in the morning after a light break-
fast. This was done by administering sufficient alcohol5 to
the individual until his blood content rose to about 0.10%.
The blood alcohol concentration was determined on the
Alcometer6 (25). Breath tests were_taken at intervals and
when the desired concentration was reached, Quick's test was
again performed and the results obtained with the two types

of individuals compared. The data are shown in Table VII.

 

5Kentucky Tavern. Bottled in bond by the Glenmore Distillers
Company, Owensboro, Kentucky.

6Alcometer, manufactured by Alfred Bicknell Associates,

Cambridge, Mass.

-20...

 

 

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SUMEARX

1. A study of oxygen consumption of liver slices from
rats previously on water, 5% ethanol and 10% ethanol showed
highest values for group receiving water and lowest for the

10% ethanol group.

2. A study of the alcohol content of tissues from the
above animals, three hours after administration of three
grams of ethanol per Kilogram of body weight, showed such

variations as not to permit definite conclusions.

5. A study of alcohol "tolerant" and "non-tolerant“ sub—
jects showed that the latter had a significantly greater
capacity to form hippuric acid after the alcohol con—

centration in the blood reached approximately .10%.

-22...

DISCUSSION

The results shown in Table III pertaining to liquid con—
sumption (water, 5% alcohol, and 10% alcohol) in this eXperi—
ment agree with those obtained by Richter (9). The animals
receiving the five per cent alcohol solution exhibited the
greatest amount of liquid consumption, followed by those on
the ten per cent level, and lastly by those on plain water.
Moreover, food consumption also coincides with his results
inasmuch as those on the ten per cent alcohol diet ate less
than the controls. Because of the variability of those on the
five per cent level, no definite conclusion can be drawn.

The metabolic rate of the liver, as determined by the
manometric Warburg technique, seems to indicate that the con—
trol animals maintained the greatest oxygen consumption, '
followed by those on the five per cent diet, with the ones on
the ten per cent diet the least. However, because of the lapse
of time between the slicing of the tissues and the actual per—
formance of the Warburg determinations, this may not be the
actual case. At the time of slicing the tissues, it would
appear that all of them had the same alcoholic concentrations
since each animal was administered the same dosage of alcohol
per 100 grams of body weight. However, during the lapse of

time between slicing and the measurement of respiration, it

-25...

may be that, in the case of the animals that had developed
tolerance, the tissues may have oxidized the alcohol almost
to completion. Whereas in the non—tolerant animals a slower
rate of oxidation occurred. Therefore, it is possible that
after a period of time, the tissue with the slower rate of
oxidation would appear to have the greatest rate of oxygen
consumption in the Warburg apparatus.

From the over~all alcohol concentrations of the tissues
as shown in Table V, there can be no definite conclusions
drawn because of the variation in values found. However,
using the average concentrations from Table VI and calcul—
ating their ratios, slight differences are noted. In Table
VIII the concentration of the blood (from Table VI) was
taken as unity, and the ratios of the alcohol concentrations

of the tissues of their respective groups were calculated.

TABLE VIII
COMPARISON RATIOS

The ratios were obtained by using the data from Table VI
and using blood as unity.

 

 

Blood Liver Kidney
CONTROL
1.0 1.64 2.01

5% ANIMALS

 

1.0 1.90 2.59
10% ANIMALS
1.0 1.59 2.51

_ 24 _

Here it may be seen that the ratio of the alcohol con—
centration of the control's liver to the five per cent to
the ten per cent is 1.64 : 1.90 : 1.68, respectively. The
ratio at the five per cent level is greater than that of
the ten per cent and the control is the smallest. Moreover,
assigning a value of one to the concentrations of the
tissues in the control animals (1. e. blood 0.714 = 1;
liver 0.1167 = l ; and kidney 0.1450 = 1), it may be seen
that the respective ratios for blood, liver, and kidney in
the ten per cent animals are 0.817 : 0.857 : 0.94 and 0.711
0.822 : 0.845 respectively for the five per'cent animals.
Hence there is a slight decrease in the alcohol concentrat—
ions—the controls being highest and the five per cent

animals lowest. The data are shown in Table IX.

TABLE IX
COMPARISON RATIOS

The ratios were obtained by using the data from Table VI
and using the control as unity.

 

 

 

Blood Liver Kidney
993258;
1.0 ~ 1.0 1.0
éZgANIMALS
0.711 0.822 0.845

10% ANIMALS

 

0.817 0.857 0.94

 

-25...

In the experimental work with the human subjects, there.
is a definite indication that the "tolerant" individuals were
less capable of forming hippuric acid than the "non-tolerant"
after the administration of alcohol. It may be postulated that
the "tolerant“ subjects are more concerned with oxidizing the
alcohol than with the formation of hippuric acid from the
benzoate, whereas the "non—tolerant" individual is primarily
concerned with the formation of hippuric acid, and consequently,
does not oxidize the alcohol as rapidly. It may be speculated
that a "tolerant“ individual has, as a primary function, "eds
ucated" his liver to oxidize the alcohol, and then to carry on
its other tasks. In the case of a "non—tolerant" person the
reverse appears to be true. Therefore, using this as a
criterion, the hippuric acid liver function test "Quick's test)
might be further examined for use as a medico—legal test to
distinguish between "tolerant" and "non—tolerant" individuals.

The main disadvantage to the Hippuric Test for Medioc—
Legal purposes is that the time required for its determination
is too long. However, there is a possibility that the Sulfo—
brom0phthalein (Bromsulfalein) test*, (28) which requires a

much shorter time, might be investigated for this purpose.

 

“The time required to run this test depends upon the amount
of dye injected. A 5 mg. dose requires from 45 to 60
minutes. A 2 mg. dose requires 20 minutes.

- 26 _

The principle of this test involves the injection of sodium
sulfobromophthalein into the blood stream. The dye is re—
moved by the liver and excreted in the bile within a short
time after injection. The degree of dye retention in the
blood is then measured by a colorimeter method. Inasmuch as
this particular test was not investigated for this purpose
in tnis paper, the author regrets the unavailability of any

statistical data concerning such.

- 27 _

11.
12.
15.
14.
15.
16.

17.
18.

BIBLIOGRAPHY

Lundsgaard, B., Skand. Arch. Physiol. 22, 56—7,(1957)
Dontoheff, L., Compt. rend. soc. biol. 126, 462—4, (1957)
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Mirsky, I. Arthur and Nelson, N., Am. J. Physiol. 127,
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LeBreton, E., Compt. rend. soc. biol. 122, 564—5 (1956)

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Newman, H. W. and Card, J., J. Nervous Mental Disease §§,
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Newman, H. N., Quart. J. Studies Alc., 2, 455-65, (1941)
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Remington, C. and Lietner, Z. A., Lancet, 249I 494-6 (1945)
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~28-

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Harger, R. V., Hulpieu, H. R., and Lamb, E. B. J. Biol.
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Umbreit, W. W., Buris, R. H., and Stauffer, J. P.,
manometric Technique in Tissue Hetabolism Minneapolis,
LLinn., J. F. Burgess Publishing Co. (1949)

 

 

 

 

Hepler, 0. E., Manual of Clinical Laboratory Diethods,
Springfield, I11,, Charles Thomas (1949)

Bennet, W. B., A Comparative Stu_y of the Reliability of

_

Several Chemical Tests for Ethyl Alcohol Intoxication in
Humans, M. S. Thesis, hichigan State College, (1950)

 

 

 

Todd, J. C. and Stanford, A. H., Clinical Diagnosis by
Laboratory Methods, Philadelphia, Pa., W. B. Saunders 00.
p. 118, (1940) -

 

Harger, R. N., Lamb, 0. B., and Hulpieu, J. Am. Med. Assoc.,

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Todd, J. C. and Stanford, A. H., Clinical Diagnosis by
Laboratory Methods, p. 112, Philadelphia, Pa. W. B. Saunders
00., (1940)

 

 

 

 

 

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