Download Determination of True Specific Activity of

Clinical Science (1982) 63,25 1-255
25 1
Determination of true specific activity of superoxide dismutase
in human erythrocytes
TAKESHI SAITO, KEIZO ITO, MASAAKI KURASAKI, SHUN FUJIMOTO,
HIROSHI KAJI AND KAZUO SAITO
Department of Environmental Medicine, Graduate School of Environmental Science, Hokkaido University, Japan
(Received I February 1982; accepted 20 April 1982)
Summarv
1. Activities, contents and true specific activities of superoxide dismutase (SOD) were
determined in human erythrocytes of 105 normal
healthy subjects.
2. The mean SOD activity assayed by the
inhibition of xanthine autoxidation was 11-0 x
lo3units/g of haemoglobin (Hb).
3. The mean SOD content assayed by an
immunochemical method was 456 pg/g of Hb.
4. Both activity and content of SOD showed
normal distributions, and no significant variations with regard to sex and age were detected.
5. A highly positive correlation between activities and contents of SOD was observed in
normal healthy subjects ( r = 0 . 7 7 , P < 0.001).
6. True specific activity was calculated from
levels of act&ity and content of SOD. The mean
true specific activity of SOD in human erythrocytes was 2 3 . 7 unitslpg of SOD.
7. There was no significant difference in true
specific activity between young and old subjects.
Key words: superoxide dismutase, human
erythrocytes, true specific activity, sex and aging.
Introduction
Superoxide dismutase (SOD) (EC 1.15.1.1)
catalyses the dismutation of superoxide anion to
Oxygen and hydrogen peroxide*Its
Correspondence: Dr Takeshi Saito, Department of
Environmental Medicine, Graduate School of
Environmental Science, Hokkaido University, Nishi-5,
Kita- 10, Kita-ku, Sapporo 060, Japan.
is considered to serve as a cell defence reaction
against the potentially harmful effects of superoxide anions generated by a wide variety of
biological reactions. Since the significance of this
enzyme was described by McCord & Fridovich
[ 1I, there have been many studies on its structure,
properties and mode of action [2, 31. Recently,
this enzyme has also received attention in the field
of practical medicine. In spite of its biological
significance and the clinical importance, fundamental investigations on SOD in erythrocytes
have not yet been performed in detail. In this
report, the activity, content and true specific
activity of SOD in human erythrocytes were
simultaneously determined in 105 normal healthy
subjects and the applicability of true specific
activity of SOD is discussed.
Materials and methods
Experimental subjects and their blood samples
Blood samples of 124 normal healthy subjects
were drawn b y a heparhized vacuum syringe
from cubital veins in the morning. Whole blood
was separated by centrifugation into blood
plasma -and erythiocyte fractions. Erythrocyte
fractions were washed with saline three times and
stored in a freezer at -8OOC until examination.
These subjects were the
of
workshops, nurses of some hospitals and the
members of a home for the aged; they were
healthy and had not been taking m y medications
within 7 days before the time of blood sampling.
The Hb content of each haemolysate was
measured cyanmethemoglobin method [41.
0143-5221/82/09025 1-05%01.50 @ 1982 The Biochemical Society and the Medical Research Society
Takeshi Saito et al.
252
Determination of SOD activity
Inhibition of SOD by cyanide
SOD activity in red-blood-cell lysates was
assayed in chloroform/ethanol extracts by the
method described by Winterbourn et al. [5l with
some modifications. The assay of activity was
basically the same as that described by
Beauchamp & Fridovich [61, and SOD activity
was determined on logit paper adopted by
Kobayashi et al. [71. The assay was based on the
inhibition of the conversion of Nitro Blue
Tetrazolium by SOD into a blue tetrazolium salt,
mediated by superoxide radicals which were
generated by xanthine oxidase. The assay was
performed in 3 mi of sodium carbonate buffer (50
mmol/l; pH 10.2) containing xanthine (0-1
mmol/l), Nitro Blue Tetrazolium (25 pmol/l) and
xanthine oxidase (5-8 nmol/l). The amount
required to inhibit the rate of reduction of Nitro
Blue Tetrazolium by 50% was defined as 1 unit of
activity. The assay was performed at 25°C and
the rate of reduction was followed at 560 nm with
a Hitachi (Tokyo, Japan) model 200-20 spectrophotometer.
Cyanide was added to samples at various
concentrations (0.01-0.1 mmol/l); these samples
were incubated for 30 min at 25"C, and then
assayed by the two methods described above.
Determination of SOD content
Human erythrocyte SOD was purified by the
method described by McCord & Fridovich [ 11.
This preparation was confirmed to be homogeneous by polyacrylamide-gel electrophoresis.
For raising antiserum in rabbits, the purified
SOD was injected subcutaneously as a waterin-oil emulsion in Freund's complete adjuvant,
and it was boosted 4 times, once a week. The
amounts of purified SOD used for immunizing
the rabbits were 1.6 mg per animal. The
specificity of the antiserum obtained against
cytoplasmic SOD was checked by the agar-gel
double-diffusion test. This antiserum was used for
the present investigation. SOD content was
determined by the immunodiffusion method
described by Mancini et al. [81. SOD content was
assayed on simple lysates of erythrocytes. A
standard curve was obtained with purified SOD.
Results
Determination
erythrocytes
of SOD activity in human
SOD activities of human erythrocytes were
determined in 124 normal healthy Japanese aged
from teens to eighties. SOD activity showed a
normal distribution, and no significant variations
were detected with regard to sex and age (Figs. 1
and 2). Mean SOD activity was 11.0 x lo3
units/g of Hb, with SD 1.44 x lo3 units/g of Hb.
Determination
erythrocytes
of
SOD content in human
SOD contents were determined in the same
samples (104 samples out of 124) as those used
for the determination of SOD activity. SOD
content also showed a normal distribution, and
no significant variations were detected with
regard to sex and age (Figs. 1 and 2). Mean SOD
content was 456 pg/g of Hb, with SD 45.7 pg/g
of Hb.
Relationships between SOD activity and SOD
content
A highly positive correlation was observed in
normal healthy subjects between SOD activity
and SOD content ( r = 0.77, P < 0.001) (Fig. 3).
Determination of SOD true spec@ activity
SOD true specific activities were determined in
the same samples (n = 105) as those used for the
determination of SOD levels. The mean true
specific activities were: male 23-4 k 0.29, female
24.1 & 0.31, and in total 23.7 ? 0.22 unitslpg of
SOD (means k SE) (Table 1).
Determination of SOD true spec@ activity
True specific activity means the activity per pg
of enzyme. We estimated it from the values of
activity and content of SOD by the following
calculation:
SOD true specific-activity unitslpg of SOD
SOD activity (units/g of Hb)
-
SOD content (pg/g of Hb)
Discussion
The average value of SOD activity (+SD)
determined by the method described was 11.0
(k1.14) x lo3 units/g of Hb. This method has
such a high sensitivity and specificity that we can
measure small changes in SOD value. This
method therefore seems to be more suitable for
clinical work than those reported previously [ 11.
Human erythrocyte superoxide dismutase
Female
Mean 1 1 1
1
10'unitslgof
25 3
Hb
Total
Mean 4 5 6 p g l g o f
so
Hb
45 7
U
3
e
L
0
"5
10
10
' x SOD activity
0
15
300
500
700
SOD content ( p g / g of Hb)
( u n i t d g o f Hh)
FIG. 1. Distribution of the activity (a) and content (b) of superoxide dismutase (SOD) in human
erythrocytes of normal healthy subjects. The means, standard deviations and numbers of subjects
( n )are shown.
..
..
m
.-m
1
..-"
c
3
x
:
1..
y
r
m
22 5x1213
017
P<OOOl
ci
X
0
I
I
I
,
1
1
1
I
10-1 9 20-29 30-3940-49 50-59 60-69 70-79 80-89
I
10
'x
SOD activity ( u n i t d g of Hh)
FIG.3. Correlation between SOD activity and SOD
content in human erythrocytes of normal healthy
subjects ( n = 105).
lb!
500
i
Ef420
0
I
I
I
I
I
10-19 20-2930-3940-4950-59
I
l
l
60-6970-7980-89
Age group (years)
FIG. 2. Superoxide dismutase (SOD) activity ( a ) and
content ( b ) in human erythrocytes of normal healthy
subjects in each age group: 0 , male, 0 , female. The
means and standard errors are shown. Black points in
parentheses are values obtained in one subject.
The average value of SOD content (456 pg/g of
Hb) in the present paper is very close to that
(46 1.4 pg/g of Hb) determined by Michelson et
al. 191 and that (500 pg/g of Hb) by Winterbourn et a/. 151, but it is lower than that (700
pg/g of Hb) reported by Stansell & Deutsch 1101
and that (850 pg/g of Hb) by Del Villano &
Tischfield I 1 1I.
In our experiments, we used packed red cells
and we express both activity and content of SOD
per g of Hb. In some experiments previously
reported 112, 131, whole blood was used for the
assay of blood cell enzymes. This procedure does
not seem to provide accurate values, since H b
Takeshi Saito et al.
254
TABLEI . True specific activity of superoxide dismutase in human erythrocytes of normal healthy
subjeets in each age group
Mean values f SE; range and numbers of subjects (n) are shown. *: Mean values, range and
numbers of subjects (n) are shown.
True specific activity (unitdpg of SOD)
Male
10-19
20-29
30-39
40-49
50-59
60-69
70-79
8&89
23.2 k 1.54
23.2 f 0.58
25.4 ?r 0.55
23.0 f 0.53
22.9 f 0.74
24.0
24.0 f 0.79
24.0
Total
23.4 f 0.29
Female
( n = 6)
( n = 14)
(n = 6)
( n = 14)
( n = 11)
( n = 2)
(n = 5)
( n = 2)
(16.0-26. I )
(19.0-26, I )
(23.9-27.7)
(1 7.7-25.2)
(17.9-26.8)
(23.2-24.8).
(22.0-25.7)
(22.4-25.6)'
\
-
?-
E
c
50
m
.-x
.->
I
4
41
1
0 -1
0.bl
24.1 + 0 - 3 1
23.7 ? 0 . 2 2
100'
0
+
( n = 60)
Grand total
8
-g
23.8
24.6 k 0.39
24.7 ?r 0 . 3 9
23.0 f 0 . 8 3
23.2
26.2
23.8 0.39
25.4
ICN-l (rnmolil)
FIG. 4. Inhibitory effects of cyanide on superoxide
dismutase activity and content in human erythrocytes. Activity (0)and content (0)are expressed as
percentages of that in controls without cyanide.
Cyanide was added to samples, and they were
incubated for 30 min at 25OC, and then assayed by the
two methods described in the text.
contents may affect levels of enzymes. Recently,
Fitzpatrick et al. I141 showed a direct relationship between catalase and Hb levels and indicated
that whole blood catalase was of no value in
diagnosis and monitoring of disease. Tudhope
[151 also showed that the effect of Hb concentration on the catalase values was negated by
the use of packed red cells adjusted to constant
(21.9-26.2)
(22.9-27.4)
(18.6-25.7)
(20.6-25.9)'
(17.4-27.0)
(24.5-26.8)'
(n = I )
( n = 10)
( n = 9)
( n = 10)
( n = 4)
( n = 1)
(n=6)
( n = 4)
( n = 45)
( n = 105)
Hb content for catalase estimation. As to the
determination of SOD levels, since Hb itself is
understood to be the principal source of superoxide anion, we took Hb contents into consideration and we expressed SOD levels in terms
of Hb concentration. But the use of true specific
activity seems to be ideal, since it is not
influenced by the effect of Hb.
Reiss & Gershon [16] demonstrated a
considerable decrease in SOD specific activity in
the liver, a very small decrease in specific activity
in the heart, and no decrease in the brain, in aged
rats and mice. Furthermore, they also showed
that the true specific activity of the 'old' cytoplasmic SOD, i.e. obtained from livers of old rats,
amounts to about 40% of the 'young' enzyme in
purified enzyme fractions [ 171. In human
erythrocytes, we confirmed no significant difference in true specific activity between young
and old subjects.
Fig. 4 showed the effect on SOD activity and
content after the addition of KCN to the sample.
With KCN at 0.1 mmol/l approximately 90% of
SOD activity was inhibited, while the content of
SOD determined by our method was unaffected.
Some red-cell enzymes lose their activity in
diseases despite normal enzyme contents. Shapira
et al. [ 181 discovered enzymically inactive carbonic anhydrase B in red blood cells in a family
with renal tubular acidosis. Our normal values for
SOD true specific activity in erythrocytes could
be applied to the study of such inactive forms of
the enzyme in various diseases.
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