CLIN.CHEM.23/9, 1602-1608 (1977)
pH and the Activity of PorphobilinogenSynthase in Human Blood
Momoko Chiba, Akiko Tashiro, and Masakazu Kikuchi
For determination
of the activity of this enzyme (EC
4.2.1.24) in blood, pH dependency was evaluated by using
whole blood and washed erythrocytes. The effects of assay
methods, original and final pH, and use of sodium or pc-
tassium phosphate buffer were examined. The pattern of
pH dependency differed for whole blood and washed
erythrocytes, whether blood from normal or lead-exposed
subjects was examined. The pH optimum for the activity
in erythrocytes from lead-exposed subjects was the same
as for the normal persons if the concentration of lead in
blood did not exceed 80 g/100 g of whole blood. Hemolyzing blood by placing it on solid CO2 decreases the activity.
AdditIonalK.yphrasei: trace elements
occupational
p51,
or environmental
an enzyme
hazards
‘
.
toxicology
lead poisoning
in the heme synthetic
pathway,
is
widely distributed
in plants, animals, and microorganisms. PS in mammalian
erythrocytes
is extremely
sensitive to inhibition by lead. In 1972, Nikkanen et a!.
(1) reported a difference in optimum pH for PS activity
between
blood specimens
from lead-exposed
and
unexposed humans. This finding was later confirmed
(2). We found that this was true only if whole blood, not
washed erythrocytes,
from moderately
lead-exposed
subjects was the analytical sample. In erythrocytes
of
highly exposed subjects, the concentration
of lead in
blood exceeded 80 sg/100 g, and the optimal pH for
activity of the enzyme had shifted from 6.8 to 6.0. We
assumed, in this case, that this was only an apparent
shift of pH optimum, resulting from the activity at pH
6.8 being decreased while that at pH 6.0 remained unchanged.
Here, we have further examined what extent of lead
exposure causes the apparent optimum pH of blood PS
activity to shift, and also the effects on blood PS activity
of differences
in assay method and composition
of
phosphate buffer.
Department
of Hygiene,
Juntendo
University
School
Hongo, Tokyo, 113, Japan.
‘Nonstandard
abbreviation used: PS, porphobilinogen
(5-aminolaevulinate
dehydratase, EC 4.2.1.24).
Received Mar. 31, 1977; accepted May 14, 1977.
1602 CLINICALCHEMISTRY,Vol. 23. No. 9, 1977
of Medicine,
synthase
Materials
Subjects
Normal human blood: Laboratory personnel who had
had no occupational or other unusual contact with lead
voluntarily supplied blood specimens.
Blood from lead-exposed
subjects: Blood specimens
were offered voluntarily from male workers engaged in
soldering in a canning factory, annealing with molten
lead in a steel-rope factory, or smelting in a refining
factory. These blood samples were classified into three
groups: low exposure (<40 Mg/100 g), moderate exposure, and high exposure (>80 g/100 g).
Whole-blood samples consisted of heparinized blood
from the antecubital vein. Samples of washed erythrocytes were derived from whole blood that was washed
and adjusted to its original hematocrit value with isotonic saline.
Hemolysis methods: To produce hemolysis, we either
(a) placed small glass tubes containing 0.2 ml of whole
blood or erythrocyte samples in a jar containing solid
CO2 for at least 30 mm, or (b) added whole blood or
erythrocyte sample to 6.5 volumes of distilled water.
Methods
Apparatus
pH/blood
gas analyzer
(Model 313, Instrumen.ation
Lab., Lexington, Mass. 02173). The pH of the sample
containing protein was measured with this apparatus.
pH-meter.
A common pH-meter with a glass electrode was used to measure the pH of the other samples,
at room temperature.
Spectrophotometer.
Absorbances
were measured
with a Model 139 spectrophotometer
(Hitachi-Beckman, Tokyo, Japan).
Reagents
Sodium phosphate
buffer. Prepare 0.2 mol/liter solution of dibasic sodium phosphate (Na2HPO4.12H20)
by dissolving 7 1.634 g in a liter of water, and of monobasic sodium phosphate (NaH2PO4’2H20) by dissolving
31.202 g in a liter of water. Mix these two solutions in
Table 1. Summary of Assay Methods for Blood PS Activity
lemolyzed
Phosphate
buff er
Concn,
mol/llt.r
WA-,
Method
ml
Composition
A
A’
Solid CO2
0.2
K + Na2
Na + Na2
B
B’
Distilled
Na + Na2
K + Na2
water,
Vol,
ml
0.2
0.1
0.2
0.5
1.5
Incubation
Totalvol.
Method
ml
A, A’
0.4
B, B’
2.5
Trlchtoroacstlc
OrigInal
Blood vol.
ml
mixture
Substrateconcn,
mmol/llter
0.2
0.2
2.5
4.0
acid concn
Final
A, A’
50 g/Iiter
42 g/liter
B, B’
100 g/Iiterb
29 9/liter
10 mmol ALA6!
liter buffer,
0.1
20 mmol ALA!
liter water,
0.5
Buffer concn,
mmoi/IIIer
100
40
EhrIIch reagent
‘r
a.....
.
ml
CH3COOH
HCIO4, 11.6 mol/liter
HgCI2, 0.2 mol/liter
concd. HCI
42.8
CH3COOH
30
16
50
HCIO4, 11.6 mol/liter
Diluted with CH3COOHto
b
Substrate
aclutlon,
ml
7.7
2.5
2.5
ALA = 5-aminolaevulinlc acid.
Plus Hgci2, 0.1 mOl/Iltor.
suitable proportions
as to prepare solutions with pH
from 5.6 to 7.6 in steps of 0.2 pH unit.
Potassium
phosphate
buffer. Prepare 0.5, 0.2, 0.1,
and 0.05 mol/liter solutions of monobasic potassium
phosphate (KH2PO4) and of dibasic sodium phosphate
(Na2HPO4.12H20),
respectively. Mix the two solutions,
each pair of the same concentrations,
in suitable proportions to make solutions with pH from 5.6 to 7.6, in
steps of 0.2 pH unit.
Buffered
substrate,
10 mmol/liter.
This substrate
solution was prepared for methods A and A’, mentioned
later. Weigh out each 8.4 mg of 5-aminolevulinic
acid
hydrochloride
(Daiichi Pure Chemical Co., Tokyo,
Japan) into each of 11 test tubes, and dissolve it in 5 ml
of each 0.2 mol/liter potassium
phosphate
buffer of
different pH (5.6 to 7.6). The 0.5,0.1, and 0.05 mol/liter
buffers were also used in the experiment
on effect of
buffer concentration.
Aqueous
substrate,
20 mmol/liter.
This substrate
solution was prepared for methods B and B’, mentioned
later. Dissolve 335 mg of 5-aminolevulinic
acid hydrochloride in 100 ml of water; the resulting pH should be
3.15.
Trichloroacetic
acid solution,
50 g/liter.
Trichloroacetic
acid containing
mercuric
chloride
reagent.
To 2.715 g of mercuric chloride (HgC12) dis-
solved in 40 to 50 ml of warm water, add 10 g of tnchloroacetic acid and dilute to 100 ml.
Modified
Ehrlich reagent A. This mixture was used
in methods A and A’. Mix 672 ml of glacial acetic acid,
120 ml of 11.6 mol/liter perchloric acid, 40 ml of 0.2
mol/liter mercuric chloride, and 40 ml of concentrated
hydrochloric acid. This may be stored at 4 #{176}C.
Immediately before use, dissolve 1 g of p -dimethylaminobenzaldehyde in 55.5 ml of the mixture, which contains
1.6 mol of perchloric acid per liter.
Modified
Ehrlich reagent B. This mixture was used
in methods B and B’. Dissolve 1 g of p-dimethylaminobenzaldehyde
in about 30 ml of glacial acetic acid, add
16 ml of 11.6 mol/liter perchloric acid, and dilute to 50
ml with glacial acetic acid. This contains 3.7 mol of
perchloric acid per liter.
Procedure
Four kinds of methods for assay of activity of PS in
blood are shown in Table 1.
Method
A. The incubation
mixture consisted of 0.2
ml of hemolysate
a (see above), 0.1 ml of potassium
phosphate buffer, and 0.1 ml of buffered substrate solution.
Method
A’. This is a modification
of method A in
which sodium phosphate buffer is used instead of potassium phosphate buffer.
Method
B. This method was described in previous
papers (1,2). Incubation mixture consisted of 1.5 ml of
hemolyzate b (see above) from 0.2 ml of whole blood or
erythrocytes,
0.5 ml of sodium phosphate buffer, and
0.5 ml of aqueous substrate solution.
Method
B’. This is a modification
of method B in
which potassium phosphate
buffer is used instead of
sodium phosphate buffer.
CLINICALCHEMISTRY,Vol. 23, No. 9, 1977 1603
Table 2. RelatIonship between pH of Original Buffer and pH In Each Step of the Assay Procedure a
Original
buffer
OrIginal
pH of
buffer
concn,
mol/Ilter
0.2
a
pH of
blood
plus
buffer
pH of
substrate
solution
7.8
7.54
7.4
6.8
6.4
6.0
5.6
7.12
6.50
6.14
5.60
4.70
pH of Incubation
mixture
7.59
7.39
0 tIm.
7.48
7.28
6.98
6.78
6.64
6.62
6.80
6.51
6.37
6.31
PS
actIvity
(A)
After
60 mln
7.44
7.25
.258
.331
6.81
6.55
6.34
6.31
.429
.374
.325
.294
Whole blood and method A’ were used.
All of the samples were incubated for 1 h in a water
bath set at 38 #{176}C
with an accurate thermoregulator.
After deproteinization
with trichloroacetic acid reagent,
and centrifugation,
3 ml of modified Ehrlich reagent A
or B was added to 1.0 ml of the supernate. After 15 mm,
the absorbance was measured at 556 nm. When a buffer
solution containing potassium was used, a white precipitate formed on addition of Ehrlich reagent at pH
below 6.8; when this happened, A
was measured after
centnifugation, to obviate any effect of the precipitation
on absorbance values.
Each measurement
was carried out in duplicate, and
the mean value was used.
Determination
of lead concentration
in blood. The
method described by Hessel (3) was used.
--.
0.8
0.6
The dilution factor in methods A and A’ for hemolysate (a) was 48, and that in methods B and B’ for hemolysate (b) was 70. In comparing the values from both
methods of hemolysis, enzyme activity was represented
by tmol porphobilinogen’(ml
erythrocyte)1.h1,
A556
being multiplied by 77/Ht in methods A and A’ and by
112.9/Ht in the other methods, where Ht is the hematocrit value and numerical values are obtained by use of
the molar absorbance
of porphobilinogen,
6.2 X 10
liter.mol’.cm’
and each dilution factor.
Results
Effect of Concentration
of Buffer Solution
First, the molar concentration
of phosphate buffer
(0.05,0.1,0.2, or 0.5 mol/liter) was examined in relation
to pH and PS activity. Table 2 shows the pH at each
step and the PS activity, whole blood from normal
subjects and method A’ being used. At all molar con-
(a)
1 .0
Calculations
U-
-
-:-
#{149}
.y
#{149}7C..
0
,‘
4,
0.4
0.2
I
--
5.6
6:0
.
6.4
68
7.2
Original
pH
(a)
7.6
>,
5
>‘
0.3
0.2
aI-
1 .4 (b)
E
0.1
1 .2
5.6
1 .0
6.4
6.8
6.0
6.4
6.8
7.2
Original
7:6
pH
0
0.8
E
#{149}
(b)
0.3
0.6
0.4
>
4.,
-
0.2
5.6
4.)
6.0
6.4
6.8
7.6
7.2
Original
pooled blood
(a) methods A and A’, (b) methods B and B’. U. whole blood; 0, washed erythrocytes; -, sodium phosphate buffer; - - -. potassium sodium phosphate buff-
er
CLINICAL CHEMISTRY,
Vol. 23. No. 9,
1977
U)
0.2
0.1
5.6
pH
FIg. 1. Porphoblllnogen synthase activity In normal subjects
1604
6.0
0.
7.2
7.6
Original
pH
Fig. 2. Porphobillnogen synthase activity In low lead-exposed
subjects’ blood
(a) methods A and A’, (b) methods B and B’. #{149},
whole blood; C), washed erythrocytes; -. sodium phosphate buffer; - - -. potassium sodium phosphate buffer
::‘ 014(a)
(a)
0.3
a)
0.05
U-
4.’
>‘
0.1
.
I..
a
5.6
6.0
6.4
E
--
6.8
7.2
Original
5.6
I
-
-
:.---i
pH
6.0
6.4
6.8
7.2
Original
pH
0.
0
E
>‘.
0.6
(b)
U
-
0.30 (b
U
E
0.5
> 0.4
I.)
0.3
4-)
>
4-)
U)
0
#{149}
7 ?
4.)
4.)
0.2
U)
-
0
0.1
5.6
6.8
7.2
Original
pH
Fig. 3. Porphobilinogen synthase activity in moderately leadexposed subject’s blood
(a) methods
rocytes; -,
er
0.20
>.
U/
4.’
6.0
6.4
5.6
6.0
6.4
6.8
7.2
Original
pH
Fig. 4. Porphobilinogen synthase activity in high lead-exposed
subjects’ blood
(a) methods A and A’, (b) methods B and B’. U, whole blood;0. washed erythrocytes;
-,
sodium phosphate buffer; -
- -.
potassium sodium phosphate buff-
er
A and A’, (b) methods B and B’. U. whole blood; 0, washed erythsodium phosphate buffer; - - -. potassium sodium phosphate buff-
U
0.4
___#{149}
‘N.
00
>‘
centrations
examined, pH was stable before and after
incubation but, when 0.05 or 0.1 mol/liter phosphate was
used, the pH range of the incubation mixture became
narrower than that when 0.2 or 0.5 mol/liter phosphate
was used, indicating that the buffering capacity of the
two former solutions is small. Because the PS activity
was found to be higher with 0.2 mol/liter solution than
with 0.5 mol/liter solution, we chose to use 0.2 mol/liter
buffer solution throughout
further studies.
Because the original pH was not equal to the final pH,
the pH used in the following descriptions is the original
pH, for the sake of convenience, and to compare with
the results described in previous papers (1, 2).
Effect of Assay Methods
for PS Activity
PS activity was determined
by the four kinds of
methods mentioned above, and the results are shown
in Figures 1-4, respectively, for blood specimens from
normal subjects, and for those with low, moderate, or
high exposure. In these graphs, (a) represents methods
A and A’, and (b) represents methods B and B’. Over the
full range of pH examined, the activities in b were higher
than in a. At pH 6.8, the pH of maximum activity, PS
activity obtained by methods B and B’ was higher than
that obtained by other methods by about 20% in normal
whole blood and about 25% in normal erythrocytes. The
marked difference in results obtained with methods A
A’ and B B’ appears to arise from one or more of the
following differences:
(a) method of hemolysis plus
differences
in degree of dilution and whether or not
specimens were stored at low temperature
(-79 #{176}C);
(b)
,
0.2
0.1
o---#{176}_0_-a---r---o_0
,:---...
0.
5:6
7:2
7:6
Original
pH
Fig. 5. Effect of low-temperature treatment on porphobilinogen
synthase activity
‘
6:0
6:4
6:8
normalwhole blood;- - - -, low lead-exposed whole blood; U, assayed by
method D; 0, hemolyzed wIth 6.5 volumes of distilled water, exposed to solid
CO2for 45 mm, and then assayed according to method B
-,
composition
of the Ehrlich reagent; and (c) the final
concentration
of trichloroacetic
acid and whether it
contained
HgC12 or not. Results of examination
on
low-temperature
treatment are shown in Figure 5. The
blood specimens from normal and low lead-exposed
subjects were divided into two series, and one series
from each of them was kept on solid CO2 for 45mm after
hemolysis with water, while the other series was kept in
an ice bath; the PS activity of all the specimens was then
concurrently
assayed by method B. The pattern of pH
dependency
persisted, although the mean decrease in
activity was 13% in blood from normal subjects and 19%
from lead-exposure
after low-temperature
treatment.
Comparison
of PS Activity
and pH Optimum
between Whole Blood and Washed Erythrocytes
Although
PS activity of whole blood agreed with that
CLINICALCHEMISTRY,Vol. 23, No. 9, 1977
1605
Table 3. Activity Ratio (pH 6.8/6.0) In Whole Blood and Washed Erythrocytes
Whole blood
Washed erytivocytes
Method
A
A’
B
B’
Low exposure
Moderate exposure
1.45
0.87
0.63
1.30
0.89
0.59
1.74
1.24
0.51
1.63
1.27
0.70
High exposure
0.21
0.33
0.40
0.30
Normal
a
s
A
3.80
2.29
12.00
A’
B
B’
2.63
4.04
4.67
6.56
1.73
1.27
0.42
7.90
2.22
0.73
0.43
activities too low to calculate the ratio.
of washed erythrocytes at pH above 6.8, the former was
markedly higher than the latter below pH 6.6. The
specimens in which maximum PS activity appeared at
pH 6.8 were normal whole blood and all cases of erythrocytes except the specimen from high lead exposure
assayed by methods B and B’. In contrast, the specimen
in which maximum activity appeared at pH 6.0 was
whole blood from lead exposure, except for one specimen from low lead exposure. The ratio of PS activity at
pH 6.8 to that at pH 6.0 was calculated from the activities obtained from all the specimens (Table 3). This
ratio decreased with the extent of lead exposure. The
ratio calculated
from activity of erythrocytes
was
markedly higher than that of whole blood, but the difference became smaller in specimens from high lead
exposure because the activity at pH 6.0 exhibits little
change, whereas that at pH 6.8 is inhibited sensitively.
1 .0
(a)
Effect of the Presence of Plasma
As shown above, PS activity differed for whole blood
and washed erythrocytes,
especially at pH 6.6 and
below. High activity was shown in the presence of
plasma in both blood from normal persons and lead
exposure. This fact suggests that there may have been
an activating substance in the plasma or that the inhibiting effect on the activity may be due to washing of
erythrocytes. To clarify this point, we did the following
experiment.
PS activity was determined for (a) whole
blood, (b) washed erythrocytes
suspended in isotonic
saline instead of plasma, (c) erythrocytes washed twice
with cold isotonic saline and suspended in the original
plasma again, and (d) plasma exchange-erythrocytes
from normal or high lead exposure were washed separately as in c, and erythrocytes
from the normal blood
were suspended
in the lead-exposed
plasma, and
erythrocytes
from the lead-exposed
blood were suspended in the normal plasma. The lead concentration
in this lead-exposed plasma was <0.2 ig/100 g. With the
normal erythrocytes (Figure 6 a) the washing procedure
0.9
0.8
0.7
c
0.6
(a)
0.5
Subject
0.4
group
#{163}
5
0.3
.
0.2
>‘
0.3
E
0.2
,
V.’-’.-
0.1
o.1
0.
5.6
>‘
4-)
6.0
6.4
>
6.8
E
5.6
6.0
6.4
6.8
7.2
Original
pH
.4.)
>‘
4.)
7.2
7.6
Original
pH
03(b)
4.)
U)
0.
>
4.)
c
4.)
0.
U)
0.
5.6
6.0
6.4
6.8
7.2
Original pH
Fig. 6. Effect of presence of plasmaon porphobilinogensynthase
activity
(a) normal erythrocytes; (b) highlead-exposed erythrocytes. #{149}.
whole blood;
0, washed erytfrocytes plus saline; A, washed erythrocytes plus noimal plasma;
U, washed erythrocytes plus lead-exposed plasma
1606 CLINICALCHEMISTRY,Vol.
23, No. 9.
1977
?
5.6
6.0
6.4
6.8
7.2
Original
7.6
pH
Fig. 7. Relationship between porphobilinogen synthase activity
in blood from subjects exposed to various extent of lead and
original pH of buffer solution
(a) whole blood, (b) washed eiythrocytes. Method B was employed. Mean lead
in blood (jig/100g): Group A, 32; B, 38; C, 48; 0, 53; E, 85
Table 4. pH of lncubation Mixture In Whole Blood I) or Washed Erythrocytes (II)
(
Original
pH of
buffer
5.6
6.0
6.4
6.8
7.4
Moderately
Method A’
Normal blood
Method B
Method A’
I
6.31
6.37
6.54
7.04
7.48
II
5.96
6.40
6.41
6.91
7.36
I
<5.9
6.18
6.52
6.91
7.33
had no effect on activity, and addition of lead-exposed
plasma (as per d) increased the activity at pH 6.6 and
below, although it still was lower than that for treatments (a) or (b). In lead-exposed erythrocytes
(Figure
6 b) the activity was almost wholly diminished. When
the plasma was restored with the original erythrocytes,
its activity increased
some, but still was less than for
whole blood. On the other hand, when the normal
plasma was added to lead-exposed
erythrocytes
(treatment
d) the activity increased markedly at pH’s
below 6.4. Thus we think that there may be an activator
in plasma
from both normal
and lead-exposed
subjects.
Effect of Composition
of Phosphate
Buffer on PS
Activity
As is evident from Figures 1 and 2, it was of little
importance for PS activity in blood whether potassium
(in phosphate buffer) was present or not. Presumably,
difference of assay method affected the activity, as
mentioned under Effect of assay methods.
The PS activity in blood from high or moderate exposure obtained
by method B at pH’s below 6.2, however, was higher
than that obtained by using a buffer with potassium,
both in the case of whole blood and erythrocytes
(Figures 3 and 4).
Discussion
In our earlier experiment
for observing pH dependency of PS activity, we had used method A and washed
erythrocytes.
Our results, therefore, did not agree with
the results of others (1, 2), who used method B and
whole blood; the pH optimum for PS activity in blood
from lead exposure accordingly was more acidic. In the
present study, the effects of difference of assay method
was examined, and we found that the discrepancy was
due to removal of plasma. When the same sample was
assayed by methods A, A’, B, and B’, PS activity obtained by method B or B’ was always higher than that
by method A or A’. The main reason for this difference
was considered to be due to mechanical destruction
of
the enzyme molecule by ice crystals during the freezing
procedure in methods A and A’.
The relationship between pH optimum of PS activity
in blood and extent of lead exposure is shown in Figure
7. Mean lead concentration
in blood for lead-exposed
II
<5.9
6.11
6.49
6.87
7.30
I
6.28
6.40
6.74
7.01
7.42
II
6.05
6.26
6.53
6.89
7.30
Pb-exposed
blood
Method B
I
5.90
6.23
6.54
6.90
7.32
II
<5.9
6.10
6.45
6.84
7.30
subjects were 32(Group A), 38(Group B), 45(Group C),
53(Group D), and 85(Group E) tg/100 g of whole blood.
Each group consisted of three to six subjects with similar lead exposure. In Group A, maximum activity was
at pH 6.8 in spite of the fact that the activity itself was
inhibited markedly. In the whole blood from Group B,
the activity on the acidic side of neutrality was generally
but not sharply higher than that on the alkaline sidei.e., there was no remarkable
peak of activity. In the
whole blood from Groups C, D, and E, a peak of activity
was observed at pH 6.0 or 6.2, and there was no peak at
pH 6.8. We anticipated that if lead loading exceeds the
extent of that in Group B, PS activity in erythrocytes
may have a sub-peak at pH 5.8 to 6.2. In addition, under
conditions of extreme loading of lead, such as in Group
E, PS activity of erythrocytes
around pH 6.8 was inhibited further, only the sub-peak at pH 6.0 persisting.
This sub-peak may also exist in normal whole blood but
be difficult to observe because the activity in the main
peak is too high, obscuring the sub-peak.
Although the effect of various metal ions on PS activity has been discussed by many investigators,
their
results do not always agree. This is true for potassium
also. Some workers suggest that the presence of potassium ion increases PS activity (4), others claim that it
has no effect (5, 6) or that it polymerizes PS (7). We
found no effect of potassium on PS activity of normal
blood, but PS activity in blood from moderate and high
lead-exposure
may have been decreased by potassium
at the more acid pH’s.
Differences between whole blood and washed erythrocytes were: (a) in blood from normal individuals, activity of whole blood is higher than that of erythrocytes
at pH values below 6.6, and (b) in blood from moderately lead-exposed
persons, the maximum activity of
whole blood is at pH 6.0, while that of erythrocytes is at
pH 6.8. The pH values mentioned above are the original
pH of the buffer solution used; the final pH of the incubation mixture differs from that of the buffer. We
measured the pH of the incubation mixture (Table 4),
and it appears that the difference of activity with and
without plasma cannot be explained merely on this
basis. We suggest that a factor that activates PS activity
of the erythrocytes on the acidic side may be present in
the plasma. Further elucidation must await continued
experiments.
CLINICALCHEMISTRY,Vol. 23, No.
9, 1977
1607
We wish to thank Dr. Kazuteru Nitta, Dr. Yoshihisa Nishino, and
Mr. Masakazu
Kikuchi for making it possible to study the factory
workers, and Dr. Taichiro Nishima for the analyses
for lead in the
blood, all from the Departments
of Hygiene and Internal Medicine
of this University.
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