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Title
Developmental changes and polymorphism in human alcohol dehydrogenase.
Permalink
https://escholarship.org/uc/item/5p67k47p
Journal
Annals of human genetics, 34(3)
ISSN
0003-4800
Authors
Smith, M
Hopkinson, DA
Harris, H
Publication Date
1971-02-01
DOI
10.1111/j.1469-1809.1971.tb00238.x
Copyright Information
This work is made available under the terms of a Creative Commons Attribution
License, availalbe at https://creativecommons.org/licenses/by/4.0/
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University of California
Ann.
Hum.
Genet.,
Lond.
(1971),
34,
251
Printed
in
Great Britain
251
Developmental changes and polymorphism in
human alcohol dehydrogenase
BY
MOYRA SMITH, D.
A.
HOPKINSON
AND
HARRY HARRIS
M.R.C.
Human Biochemical Genetics Unit, Galton Laboratory,
University College London
In man, alcohol dehydrogenase (alcohol: NAD oxidoreductase E.C.
1.1.1.1)
occurs princi-
pally in liver, though low levels
of
activity have aIso been found in lung, kidney and the gastro-
intestinal tract (Moser, Papenberg
&
von Wartburg,
1968).
Evidence for
at
least three distinct
isozymes has been obtained by chromatography
of
liver extracts on CM cellulose (Blair
&
Vallee,
1966)
and also by electrophoresis (Moser
et
al.
1968;
Pikkarainen
&
Raiha,
1969;
Murray
&
Motulsky,
1970).
Von Wartburg, Papenberg
&
Aebi
(1965)
reported that certain individuals have an atypical
form of alcohol dehydrogenase associated with an increased level of activity. The usual and
atypical forms of the enzyme were shown to differ markedly in pH activity curves with ethanol
as substrate. The pH optimum for the usual form was found to be pH
10.8
and for the atypical
form pH
8.5.
The enzymes also differed in the relative rates at which they oxidized various other
alcohols, and in the degree of inhibition produced by various metal binding agents. On the
other hand no significant differences were observed in Michaelis constants for the substrates
ethanol or acetaldehyde
or
for the corresponding coenzymes NAD or NADH. Also the pH
activity curve with acetaldehyde as substrate
was
essentially the same for both enzymes,
having an optimum at pH
6.0-6.5.
A
simple screening test to distinguish the usual from the atypical enzyme in crude liver homo-
genates was designed (von Wartburg
et al.
1965).
This involves determining the ratio of the
activity at pH
11.0
to that
at
pH
8-8
with ethanol as substrate under standard conditions. The
usual enzyme gives
a
value
for
this ratio greater than
1.0,
and the atypical enzyme less than
1.0.
In
a
survey of
59
liver samples from different individuals in Switzerland,
12
were found to have
the atypical alcohol dehydrogenase, and in another series of
50
individuals from London,
2
were
found to be atypical (von Wartburg
&
Schiirch,
1968).
The atypical enzyme occurred in indi-
viduals varying from
16
to
82
years of age.
Pikkarainen
&
Raiha
(1967)
reported that alcohol dehydrogenase activity in liver is low
during foetal life and reaches adult levels about
5
years after birth. Changes in electrophoretic
pattern have also been noted during development (Pikkarainen
&
Raiha,
1969;
Murray
&
Motulsky,
1970).
In the earliest stages only
a
single isozyme is observed but later further iso-
zymes appear. In adult liver individual variations in the relative contribution of the different
isozymes to the total activity have been noted (von Wartburg
&
Schiirch,
1968),
but no clear
electrophoretic differences between the usual and atypical alcohol dehydrogenases as determined
by the ratio of activity at pH
11.0
and pH
8.8
were detected.
The present paper is concerned with
a
study of human alcohol dehydrogenase in which liver,
lung, kidney and intestinal material from foetuses, infants and adults has been examined. The en-
zyme has been investigated both by spectrophotometric assay at different
pH’s
and by starch-gel
252
M.
SMITH,
D.
A.
HOPKINSON
AND
H.
HARRIS
electrophoresis. The findings suggest that
at
least three distinct gene loci may be concerned in
determining the structure
of
the various alcohol dehydrogenase isozymes in man, and that they
are active to markedly different degrees in different stages of development in different tissues.
MATERIALS
AND METHODS
Foetal samples
(9-22
weeks gestation) were obtained from therapeutic abortions. Other samples
were obtained from autopsies, and were mainly from adults but some came from premature
infants and
a
few from young children. The autopsy samples were generally taken'between
12
and
48
hr. after death, the body having been refrigerated in the meanwhile. The samples if
not examined immediately were stored at
-20"
C. Care was taken to avoid repeated freezing and
thawing
of
samples since this was found to lead to a rapid decay of alcohol deliydrogenase activity.
The tissues examined in detail were liver, lung, kidney and intestine. Although traces of alcohol
dehydrogenase activity probably occur in certain other tissues, the activity was found to be much
too low to be investigated by the techniques used here.
Assay of alcohol dehydrogenase was carried out by the method described by von Wartburg
et
al.
(1965)
using ethanol as substrate. In each case an assay was carried out at pH
8.8
and also
at
pH
11.0.
For
assays of the enzyme
in
liver, the tissue was homogenized in
9
volumes of 0.1~
phosphate buffer
pH
7.0
and the debris removed by centrifugation at
3000
rev./min. for
15
min.
;
0.2
ml. of the supernatant was added to
2.8
ml.
of
the reaction mixture, giving
a
final volume of
3
ml. which contained
1.6
x
10-3~ NAD and
1.6
x
1OW2~
ethanol in
3.3
x
M
sodiumpyrophos-
phate buffer
pH
8.8
or
3.3
x
lop2
M
glycine/NaOH buffer pH
11-0.
The reaction was followed in a
Gilford spectrophotometer at
340
mp and at
25"
C.
A
blank reaction without ethanol was also
run. The enzyme activity was expressed as the change in optical density per minute per gram
of tissue. The assays on lung tissue required more concentrated extracts,
so
here the tissue was
homogenized in
2
vol. of phosphate buffer and after centrifugation
0-2
ml. of the supernatant
was used in the reaction mixture.
For electrophoresis the tissue was homogenized with an equal volume of water in the case of
liver.
For
lung, intestine and kidney the samples were generally homogenized directly without
any addition of water. After centrifugation the supernatant was subjected to horizontal starch-
gel electrophoresis the inserts being made with filter paper (Whatman no.
17).
The gel buffer
was
0.025
M
Tris/HCl
at
pH
8.6
and contained
4
x
M
NAD. The bridge buffer was
0.3
M
Tris/HCl
pH
8-6.
Suitably insulated metal cooling plates through which chilled water
(5-10"
C.)
circulated were used to control the gel temperature during electrophoresis. Electrophoresis was
either carried out for
4-5
hr.
at
12
V./cm.
or
for
16
hr.
at
5
V./cm.
Following electrophoresis the gels were sliced and stained using an agar overlay. Alcohol
dehydrogenase was detected with
a
reaction mixture containing
20
mg.
NAD,
10
mg. tetra-
zolium salt MTT,
2
mg. phenazine methosulphate and
0.1
ml. ethanol in
25
ml.
0.05
M
Tris/HCl
buffer at pH
8-6
mixed with
25
ml.
2
yo
aqueous agar
at
55'
C.
The gels were incubated at
37"
C.
for
1-2
hr.
RESULTS
(a) Activity determinations Liver
Alcohol dehydrogenase was assayed at both pH
8.8
and pH
11.0
in
222
different liver samples
using ethanol as substrate. The samples came from individuals of both sexes and covered a
Changes
in
human alcohol dehydrogenase
253
wide range of ages:
56
were from early foetuses
(9-22
weeks gestation),
37
from premature
infants and infants less than
1
year old, and the remainder were adults over
20
years of age.
Fig.
1
shows a plot of activity at pH
11.0
against activity at pH
8-8,
each point representing a
separate individual. Because of the very wide range of activities observed at both pH's
a
log. scale has been used. The points appear to be distributed into two distinct groups. In the
larger group the activity at pH 11.0 is greater than
at
pH
8-8.
In
the smaller group the opposite
is the case. Within each group there is
a
good correlation between activity
at
pH
8.8
and activity
at pH 11.
15
10
8
6
5
4
3
0
-2
v-
I,
CI
x
>
LI
.-
.-
r
:I
I
2
0.8
0.6
0.5
0.4
0.3
0.2
0.1
0
0
-*
. .
00
.
.
.*
..
..
'0
.
v
0'
0.
@o
P
:
.
.O*
.
0' 0
A
A
A
A
A
O.
tl
O
'0
0
OeO
00
0
0
.
A
A
A
A
A
AA
Adults and infants:
pH
ratio >1.0
A
Adults and infants:
pH
ratio
<1.0
0
Foetuses
.
A
I
A
I
I1111
I
I
I
I
IIIII
I I
I
0.2
0.3
0.4
0.50.6
0.8
1
2
3
456
810
20 30
40
ADH
activity at
pH
8.8
Fig.
1.
Plot
on
log.
scale
of
alcohol dehydrogenase activity (0.D.3ao/min./G tissue)
at
pH
11
against
activity
at
pH
8.8
in liver samples from
129
adults,
37
premature infmts and infants between term
and ono year and
56
foetuses. Each point represents
a
separate individual.
The samples with greater activity at pH
8.8
presumably represent those which have the
'atypical' alcohol dehydrogenase described by von Wartburg
et
al.
(1965). This interpretation
has been confirmed in several cases by determining the full pH activity curves. 'Atypical'
samples with higher activities at pH
8.8
were found to have pH optima close to this value whereas
other samples were found to have pH optima close to pH
11.0.
254
M.
SMITH,
D.
A.
HOPKINSON
AND
H. HARRIS
The
pH
activity ratio (activity at pH ll.O/activity
at
pH
8.8)
which was suggested by von
Wartburg
as
a simple discriminant between the two types of enzyme has been calculated for
each sample. The distribution
of
values of this ratio in the series of samples
is
shown in Fig.
2.
Pig.
2
(a)
shows the distribution
of
the values
of
this ratio in
166
samples obtained from adults
24
22
20
18
16
14
12
10
8-
6-
4-
=
2-
'p
-0-
c:
Y)
-
d
.-
-
-
-
-
-
-
-
-
la1
Adults
._
%
0.2 0.6
1.0
1.4
1.8
2.2 2-6 3.0 3.4
3.8
4.2
Foetuses
10
8
6
4
2
0.2 0.6
1.0
1.4
1.8
2-2 2.6 3.0
3.4
3.8 4.2
pH
activity ratio
Fig.
2.
Distribution
of
alcohol dehydrogenase
pH
activity ratios (activity at pH Il/activity at
pH
8.8)
in liver samples from
(a)
129
adults and
37
premature infants and infants between term
arid
one
yoar
of
age,
(b)
56
foetuses. Each square represents one individual.
and infants.
It
is clearly bimodal. Sixteen samples can be seen to have the atypical pH ratio
phenotype, and
150
to have the usual pH ratio phenotype. The atypical phenotype was found
in both
sexes
and over a wide
range
of ages. The youngest was in
a
premature infant of about
28
weeks gestation, and the oldest in an individual
of
90
years.
Fig.
2
(b)
shows the distribution
of
pH ratios in fifty-six samples from young foetuses
(9-22
weeks gestation). Here all the values fall into the range
of
the usual pH ratio phenotype. The