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δ-Aminolevulinic acid dehydratase activity in erythrocytes for the evaluation of lead poisoning
CLINICA
CHIMICA
ACTA
&AMINOLEVULINIC FOR
THE
319
ACID DEHYDRATASE
EVALUATION
OF
LEAD
ACTIVITY
IN ERYTHROCYTES
POISONING
SUMMARY
The S-aminolevulinic acid (ALA) dehydratase activity in erythrocytes, the urinary output of ALA, coproporphyrin and lead, and the level of lead in the blood were determined simultaneously in workers exposed to lead poisoning. The decrease of ALA dehydratase activity in lead poisoning (0.263 & 0.081 pmoles PBG/ml erythrocyte/h as compared with 0.920 & c.162 for normal value) correlates very closely ($J < 0.01) with the raised blood level of lead and urinary output of ALA, and significantly (~5 < 0.05) with the duration of exposure to lead. However, poor correlations were found between the decrease in ALA dehydratase activity and the increased urinary outputs of coproporphyrin and lead. Erythrocytes obtained from patients with other neurological and hematological disorders showed normal activity of ALA dehydratase. Reduced glutathione was effective for the recovery of the decreased ALA dehydratase activity in lead-poisoned erythrocytes ilz Go. It is concluded that the determination of ALA dehydratase activity in erythrocytes offers an excellent measure for the evaluation of lead poisoning and that administration of reduced glutathione seems to be useful for treating patients with lead-poisoning.
INTRODUCTION
There have been many reports on abnormal levels of porphyrins and porphyrin precursors in urine, serum, erythrocytes and bone marrow in lead poisoningI. They also report on elevated urinary levels of &aminolevulinic acid (ALA) and coproporphyrin which are considered essential for the diagnosis of lead poisonin@. On the other hand, there is evidence of a direct inhibitory effect of lead in vitro on ALA dehydratase314. There is also evidence of a decreased activity of the enzyme in erythrocytes obtained from patients with lead poisonir@. It has been generally agreed that increased urinary excretion of ALA ensues from the decreased activity of ALA dehydratase in erythrocytes and the amount of urinary ALA closely correlates with the severity of lead poisonin@. Therefore, estimation of the activity of this enzyme might give an earlier and more reliable indication of lead poisoning. The aim of the present investigation was to study the correlation between ALA Cl&.
Chim.
Acta,
19 (1968)
319-325
.G w G J ,::
5
42
45
52
45
32
3
4
5
6
7
Normal S.D.
9 IO II I2
mean
44 4’ 39 39 36
33
2
8
so
Age
I
~_____
Patiexf
_
paresthesia, colic
irritability,
epigastric myalgia headache,
arthralgia,
constipation,
_._
_
-~ of
loss of weight,
ab-
colic, con-
loss of
taste,
loss
abdominal
cramps,
metal
abdominal
muscle
pain,
paresthesia,
myalgia,
_
Fatigue, wrist-drop, loss of tendon reflex, constipation Fatigue, metal taste, myalgia Fatigue, arthralgia, nausea Fatigue, myalgia, nausea, numbness Fatigue, arthralgia, abdominal colic Fatigue, irritability, rnyalgia
Fatigue, colic Fatigue, weight Fatigue, numbness, Fatigue, weight Fatigue, stipation Fatigue, dominal
14.3 IA.6
1t.G
13-i 13.7 12.8
I2.0
9.9
12.4
X3.8
12.8
12.8
22.9 8.2
I4I.O 80.0 165.0 Ii6
Ij2.0
14X.0
____.~~
I.4 0.9
37.4 57.3 48.4 29.0 34.8 go.2
0.29 O.I4
2.8 6.1 3.1 2.5 6.8 2.8
18.5
7.0 4.0
29.1 4.8
1I.j
68.7 15-4
0.920 0.162
0.395 0.126 0.285
0.210
0.182
0.20s
0.2jj
0.4’2
0.230
0.2g=j
0.2Gr
0.298
ALA
DEHYDRATASE
IN LEAD
321
POISONING
dehydratase activity in erythrocytes and other parameters of lead poisoning, Further, we have compared the activity of this enzyme in various kinds of neurological and llenlatological diseases resembling lead poisoning. In addition, the effect of glutathione on the activity of ALA dehydratase was studied in patients with lead poisoning in v&o. SUBJECTS
Twelve patients with clinical evidence of lead poisoning (Table I) and seventeen workers exposed to lead for various lengths of time, but without any clinical symptoms of lead poisoning were studied. Sixteen of them had been working in a metal mine, being chiefly engaged in lead-smelting, and the remaining thirteen were employed in the lens manufacturing industry.
Standard methods were used for the determination of hemoglobin, reticulocyte count and basophilic stippling. ALA in urine was determined according to Mauzerall and Granick7, coproporphyrin in urine according to Rimingtons, and lead in whole blood and urine as described by Moncrieff et ~1.~. ALA dehydratase activity was determined by a slightly modified method of Granick and Mauzeralllo. Blood was collected with heparin and kept at o0 until incubation. The plasma was removed and the cells were washed twice with ice-cold o.S5”/$ saline. The erythrocytes were then resuspended in cold 0.85% saline to restore the original volume. After measuring the hematocrit, the resuspended erythrocytes were lysed by rapid freezing and thawing twice. Ilemolyzed erythrocytes were used as the source of the enzyme. Incubation was carried out in a mixture containing 0.3 ml of lysed erythrocyte suspension, 0.1 ml of 0.1 M phosphate buffer at pH 7.0, and 0.1 ml of 0.05 M ALA under N, gas at 37” for I h. Control tubes were identical except that they contained 0.1 ml of water in place of ALA solution. The reaction was terminated by the addition of I ml of 7 9/otrichloroacetic acid and 0.05 ml of saturated CuSO, solution. Porphobilinogen in the aliquot was determined by addition of modified Ehrlich’s reagent according to the method of Mauzerall and Granick?.
Ten workers were isolated from exposure to lead. Five of them received intramuscular injections of reduced glutathione (“Tathion”, obtained from the Yamanouchi Co.) in doses of 200 mg per day for 30 days. ALA dehydratase activity was measured before and after the injections and compared with that of five controls. RESULTS
Clinical and laboratory data of the patients with symptoms of lead poisoning are given in Table I. Despite a great variation in the clinical picture, laboratory testsespecially of lead in blood and ALA in urme-uniformly showed clearly abnormalvalues consistent with lead poisoning. Hemoglobin concentration varied from 9.9 to 14.3 g per IOO ml, with a mean of 12.6. The mean reticulocyte count was r.g’j/,, ranging Clin. C&n.. Acta, 19 (1968)
319-325
from 0.5 to 3.996. Coproporphyrin levels ranged from 4.8 to 9r.z g per roe ml urine. The activity of ALA dehydratase was markedly decreased in all patients, ranging from 10 to 40:/o of normal, with a mean of 0.263 ,umoles of porphobilinogen synthe-
I
50 .I00 Pb in BlOOd pg/K)Oml
150
Pb in Urine
pg/lOOml
Fig. I. Correlation between x2.lues for lead in blood and ALA dehydratasc activity in red blood cells (REV), AL)\ dehydratasc actix-ity is expressed as ,~tmoles of prphobilinogcn synthcsizcd per ml of packed red blood cells per hour of incubation. +z 7 IL, Y : -0.75, p < -o.or. 1:jn o. 2. Correlation between x-alues for lead in urine and AL.4 cells (IWC). II =- 12, I, = -o.& p 7 0.2.
tlehytlrntase
activity
in ~-cd blood
# 0.5r .c 0.4: ;; 0.3$
: .
l.
20.2-
.
8
G
Ghi;hkk; ALA
in Urine
.
0.1
d
10
20
30
40
COPROPORPHYRIN
mg/‘iOOml
I-ix. 3, Correlation bctneen xxlues for ALA cells (RX). ~7 : IL, Y E ---0.76, fl < 0.01.
in urine and AL.4
0.6 0.5r
2
0 0.5-
x .
.c 0.4 _
.s $ 0.4
z s 0.3. 2 7II 20.2.
l. .
.
8 y 0.3,
.
l
.
60
dchydratasc
7oi%eo
flgf100ml
activit),
in rctl blood
tlehytlratasc
activity
l.
.
.
. .
. .
$0.2. 0
.
Q O.lr
in
‘: . .
D
‘.
&
50 in Urine
I’ig. 1, Correlation between x~~lues for coproporphyrin in urine and ;\LA IIT1 blood cells (KRC). ?1= 12, Y = -o.lg*, p : non-significant.
”
. .
. .
.
..
. .
SJ 0.1Q 2 RETI:“LOCYTES
Fig. 5. Correlation blood cells (RBC).
3
4
%
of
between values for reticulocytes in blood and AL.4 n = IL, Y = 0.05, p = non-significant.
Fig. 6. Correlation between the duration of exposure in red blood cells. n = 17, Y = -0.61, $J < 0.05. Clin. C/hiv~. Acta,
5 DURATION
19 (1968) 319-325
10 EXPOSURE
15 to Pb
dehydratase
to lead and the activity
of ALA
20 Years
activity
in red
dehydratasc
ALA
DEHYDRATASE
IN LEAD
POISONING
323
sized per ml of packed red blood cells per hour of incubation.
No correlation
was found
between the number of stippled cells and the other laboratory tests. Figs. I to 5 show the correlations between the activity of ALA dehydratase and the values for lead, ALA, coproporphyrin, or reticulocyte count. The decrease of ALA dehydratase activity in lead-poisoned erythrocytes correlated very closely ($J < 0.01) with the levels of lead in whole blood and ALA in urine, showing no clear correlation with tlie values for lead in urine, coproporphyrin in urine, and reticulocyte count in peripheral blood. Table II shows the activity of AL,4 dehydratase in erythrocytes obtained from patients with various kinds of neurological and hematological diseases. Marked decrease in ALA dehydratase activity was found only in the lead-poisoned erythroTABLE
II
Iliclg,zosis (X0. Of patlmts) Normal
A LA
dehvdvnfnsr
nctioit~“*
(IL)
Pb poisoning symptoms (+) (12) Symptoms (-~) (17) As poisoning (I) CO poiwning (I) Infectious polyneuritis (3)
0.
102+
O.‘)O
_I
0.263
I 0.081*
Cl..+01 1~ o.r81*
0.794 0.967 0.930,
0.902,
Diabetic neuropsthy (2) 0.889, 0.912 Subacute myelooptico0.989, 0.826 neuropathy (4) 0.698, 0.849 Acute intermittent porphyria (2) 0.998, x.08 Dejerinc-Sottas’s disease (I) 0.879 Multiple sclerosis (2) 0.798, 0.882 Amyotrophic
lateral
sclerosis(z)
Meningitis t,uberculosa \Vilson’s disease (I) Parkinsonian syndrome
(I) (2)
* Mean -1 S.1). ** Activity of _%LA dehvdratase packed red blood cells per hour
0.771, 0.912 0.768 0.776,
~1.901
0.698
is expressed of incubation.
0.894
Subacute combined dcgcneration (r)
0.u ‘J
Dystrophia
0.891, 0.709,
musculorum
ProB. (4) Polymyositis (I) Myotonia dystrophica(r) Brain tumor (2) Spinal tumor (I) Cervical spondylosis (L) iron deiiciency anemia (3) Hemolytic ar:cmia (2) htcgaloblastic anemia(r) Hypoplastic anemia (3) Sideroachrestic
anemia
(I) :\cutc myclocytic lcukcmia (7) Chronic myclocytic leukemia (1) as /~molcs porphobilinogen
0.990 1.120
0.05’
0.881
0.890.
1.Oj.j
0.751 0.698, I.Z~O, 0.889
0.890 1.309
1.360,
I..+O.j
0.685,
0.709 0.998
C.OOj,
0.599 I.055 0.780,
0.695
0.668,
0.881
synthesized
per ml of
cytes, though only a slight difference in the activity was found between patients with symptoms of lead poisoning and workers exposed to lead without such symptoms. Fig. 6 shows the relationship between the decrease of ALA dehydratase activity and the duration of exposure to lead. Decrease of the enzyme activity was already found in workers after 3.2 years of exposure, and a close correlation was demonstrated between the length of time and the decrease of enzyme activity. Although isolation and discontinuation of the exposure to lead for a month improved ALA dehydratase activity but slightly, a statistically significant (fi < 0.05) recovery in the enzyme activity was found in workers who had received injections of reduced glutathione, as compared with those without injections (Table III). Clin. Chim. Acta,
19 (1968)
3x9-325
_ I 2
I
3
\
4 5 Mean
6 87
-‘:
Isolation
from exposure
to lead alone
S.D.
0.32
0.25
0.29
0.04
0.22
0.32
0.10
0.26
0.29
0.o.j
0.21
0.22
0.01
0.26
\
9
0.3.5
Isolation
from exposure to lead plus of reduced injections glutathione**
_‘~ 0.05
0.24
0.20 o.24
IO
0.34 0.29
Mean & S.D.
0.26 ~~ o.oj
-0.0.3
o.oq 0.39
0.37 0.30 0.46 0.37
:y- 0.0-l
0.l.j
0.06j 0.1 0.12
0.08 0.12
I 0.o.J I’. -: 0.05
* Exprcsscd as ,umoles of porphobilinogen synthesized per ml of packed red blood cells per hour of incubation. ** Reduced glutathionc was injected intramuscularly in doses of zoo mg/day for 30 days. DISCUSSIOIi
Several authors reported that increased urinary excretion of ALA appears to be closely correlated with lead poisoningr*rr9r2. Furthermore, Holmqvist13, in an investigation on 149 workers, found a good correlation between lead in the blood and exposure to lead. On the other hand, De Kretser and Waldron14 found only a poor correlation between ALA and the urinary lead output in an investigation on roe lead workers, whereas Haeger-Aronsenl found a good correlation. The present study showed that the activity of ALA dehydratase was markedly decreased in almost all the lead-poisoned erythrocytes, and that the decrease of the enzyme activity was closely correlated with the increased blood level of lead and urinary level of ALA, and with the duration of the exposure to lead. These results indicate that the measurement of ALA dehydratase activity in the erythrocytes offers a good and simple method of evaluation of lead poisoning, in view of the difficulties involved in the determination of ALA. Lead in the blood is said to be rapidly incorporated into red blood cells and probably affects ALA dehydratase 1,)~ directly inactivating the sulfhydryl groups necessary for its activitys. Considering the facts that the peripheral mature erythrocytes do not actually synthesize ALA and that the increased ALA probably originates from immature erythroid cells in the bone marrow, a good correlation between urinary ALA and erythrocyte ALA dehydratase activity seems to indicate the possible inhibition of ALA dehydratase of immature erythroid cells in the bone marrow along with that of mature erythrocytes in the peripheral blood. As can be seen in Table II, neurological and hematological disorders resembling lead poisoning could be differentiated from lead poisoning by measuring ALA dehydratase activity in erythrocytes, although this enzyme activity might not discriminate between clinically obvious cases of lead poisoning and latent ones. Lichtman and Feldman5 demonstrated the reversal of the ALA dehydratase deficit in C&n. Chim.
Acta,
19 (1968)
319-325
ALA DEHYDRATASE
IN LEAD POISONING
325
the lead-poisoned erythrocytes by preactivating the incubation mixture with glutathione. In the present study, in viva administration of reduced glutathione in workers with lead poisoning improved the decreased ALA dehydratase activity to some extent. For the recovery of ALA dehydratase activity, reduced glutathione seems to be effective in vivo as well as in vitro. ACKNOWLEDGEMENT
We thank
Drs. H. Katsunuma
and T. Suzuki
for their kind helps.
REFERENCES I B. HAEGER-ARONSEN, Stand. J.Clin. Lab. Iwest., Suppl., 12 (1960) 47. AND J. J. CHISOLM, J. Pediat, 56(1960) 759. 3 K. D. GIBSON, A. NEUBERGER AND J. J. SCOTT, B&hem. J.. 61 (1955) 618. 4 E. I. B. DRESEL AND J. E. FALK, B&hem. J., 63 (1956) 80. 5 H. C. LICHTMAN AND F. FELDMAN, J. Clin.Invesl., 42 (1963) 830. 6 A. GOLDBERG, J. A. SMITH AND A. C. LOCHHEAD, Brit. Med.J., i(Ig63) 1270. 7 D. ~M~UZERALL AND S. GRANICK, J. Biol.Chem., zig (1956) 435. 8 C. RIMINGTON, Assoc. Clin.Pathologists, Broodsheet N. S., No. 21. Nov. (1958). 9 A. A. MONCRIEFF, 0. P. KOUMIDES, B. E.CLAYTON,A. D. PATRICK,A.G.C. RENWICK G. E. ROBERTS, Arch. Diseases Childhood, 39 (1964) I. IO S. GRANICK AND D. MAUZERALL, J.Biol. Chem., 232 (1958) Irrg. II R. C. GRICCS AND J. W. HARRIS, Clin.Res., 2 (1958) 188. 12 K. CRAMI?R AND S. SELANDER, Brit.J. Ind.Med., 22 (1965) 311. 13 I. HOLMQVIST, Schri_ften Ges. Deut. Metallhiitten und Bergleute, 7 (1960) 115. 14 A. J. DE KRETSER AND H. A. WALDRON,BY~~. J. Ind. Med., zo(rg63) 35. 2 P. E. BENSON
C&z. Chim. Acta,
19 (1968)
AND
319-325
CHIMICA
ACTA
&AMINOLEVULINIC FOR
THE
319
ACID DEHYDRATASE
EVALUATION
OF
LEAD
ACTIVITY
IN ERYTHROCYTES
POISONING
SUMMARY
The S-aminolevulinic acid (ALA) dehydratase activity in erythrocytes, the urinary output of ALA, coproporphyrin and lead, and the level of lead in the blood were determined simultaneously in workers exposed to lead poisoning. The decrease of ALA dehydratase activity in lead poisoning (0.263 & 0.081 pmoles PBG/ml erythrocyte/h as compared with 0.920 & c.162 for normal value) correlates very closely ($J < 0.01) with the raised blood level of lead and urinary output of ALA, and significantly (~5 < 0.05) with the duration of exposure to lead. However, poor correlations were found between the decrease in ALA dehydratase activity and the increased urinary outputs of coproporphyrin and lead. Erythrocytes obtained from patients with other neurological and hematological disorders showed normal activity of ALA dehydratase. Reduced glutathione was effective for the recovery of the decreased ALA dehydratase activity in lead-poisoned erythrocytes ilz Go. It is concluded that the determination of ALA dehydratase activity in erythrocytes offers an excellent measure for the evaluation of lead poisoning and that administration of reduced glutathione seems to be useful for treating patients with lead-poisoning.
INTRODUCTION
There have been many reports on abnormal levels of porphyrins and porphyrin precursors in urine, serum, erythrocytes and bone marrow in lead poisoningI. They also report on elevated urinary levels of &aminolevulinic acid (ALA) and coproporphyrin which are considered essential for the diagnosis of lead poisonin@. On the other hand, there is evidence of a direct inhibitory effect of lead in vitro on ALA dehydratase314. There is also evidence of a decreased activity of the enzyme in erythrocytes obtained from patients with lead poisonir@. It has been generally agreed that increased urinary excretion of ALA ensues from the decreased activity of ALA dehydratase in erythrocytes and the amount of urinary ALA closely correlates with the severity of lead poisonin@. Therefore, estimation of the activity of this enzyme might give an earlier and more reliable indication of lead poisoning. The aim of the present investigation was to study the correlation between ALA Cl&.
Chim.
Acta,
19 (1968)
319-325
.G w G J ,::
5
42
45
52
45
32
3
4
5
6
7
Normal S.D.
9 IO II I2
mean
44 4’ 39 39 36
33
2
8
so
Age
I
~_____
Patiexf
_
paresthesia, colic
irritability,
epigastric myalgia headache,
arthralgia,
constipation,
_._
_
-~ of
loss of weight,
ab-
colic, con-
loss of
taste,
loss
abdominal
cramps,
metal
abdominal
muscle
pain,
paresthesia,
myalgia,
_
Fatigue, wrist-drop, loss of tendon reflex, constipation Fatigue, metal taste, myalgia Fatigue, arthralgia, nausea Fatigue, myalgia, nausea, numbness Fatigue, arthralgia, abdominal colic Fatigue, irritability, rnyalgia
Fatigue, colic Fatigue, weight Fatigue, numbness, Fatigue, weight Fatigue, stipation Fatigue, dominal
14.3 IA.6
1t.G
13-i 13.7 12.8
I2.0
9.9
12.4
X3.8
12.8
12.8
22.9 8.2
I4I.O 80.0 165.0 Ii6
Ij2.0
14X.0
____.~~
I.4 0.9
37.4 57.3 48.4 29.0 34.8 go.2
0.29 O.I4
2.8 6.1 3.1 2.5 6.8 2.8
18.5
7.0 4.0
29.1 4.8
1I.j
68.7 15-4
0.920 0.162
0.395 0.126 0.285
0.210
0.182
0.20s
0.2jj
0.4’2
0.230
0.2g=j
0.2Gr
0.298
ALA
DEHYDRATASE
IN LEAD
321
POISONING
dehydratase activity in erythrocytes and other parameters of lead poisoning, Further, we have compared the activity of this enzyme in various kinds of neurological and llenlatological diseases resembling lead poisoning. In addition, the effect of glutathione on the activity of ALA dehydratase was studied in patients with lead poisoning in v&o. SUBJECTS
Twelve patients with clinical evidence of lead poisoning (Table I) and seventeen workers exposed to lead for various lengths of time, but without any clinical symptoms of lead poisoning were studied. Sixteen of them had been working in a metal mine, being chiefly engaged in lead-smelting, and the remaining thirteen were employed in the lens manufacturing industry.
Standard methods were used for the determination of hemoglobin, reticulocyte count and basophilic stippling. ALA in urine was determined according to Mauzerall and Granick7, coproporphyrin in urine according to Rimingtons, and lead in whole blood and urine as described by Moncrieff et ~1.~. ALA dehydratase activity was determined by a slightly modified method of Granick and Mauzeralllo. Blood was collected with heparin and kept at o0 until incubation. The plasma was removed and the cells were washed twice with ice-cold o.S5”/$ saline. The erythrocytes were then resuspended in cold 0.85% saline to restore the original volume. After measuring the hematocrit, the resuspended erythrocytes were lysed by rapid freezing and thawing twice. Ilemolyzed erythrocytes were used as the source of the enzyme. Incubation was carried out in a mixture containing 0.3 ml of lysed erythrocyte suspension, 0.1 ml of 0.1 M phosphate buffer at pH 7.0, and 0.1 ml of 0.05 M ALA under N, gas at 37” for I h. Control tubes were identical except that they contained 0.1 ml of water in place of ALA solution. The reaction was terminated by the addition of I ml of 7 9/otrichloroacetic acid and 0.05 ml of saturated CuSO, solution. Porphobilinogen in the aliquot was determined by addition of modified Ehrlich’s reagent according to the method of Mauzerall and Granick?.
Ten workers were isolated from exposure to lead. Five of them received intramuscular injections of reduced glutathione (“Tathion”, obtained from the Yamanouchi Co.) in doses of 200 mg per day for 30 days. ALA dehydratase activity was measured before and after the injections and compared with that of five controls. RESULTS
Clinical and laboratory data of the patients with symptoms of lead poisoning are given in Table I. Despite a great variation in the clinical picture, laboratory testsespecially of lead in blood and ALA in urme-uniformly showed clearly abnormalvalues consistent with lead poisoning. Hemoglobin concentration varied from 9.9 to 14.3 g per IOO ml, with a mean of 12.6. The mean reticulocyte count was r.g’j/,, ranging Clin. C&n.. Acta, 19 (1968)
319-325
from 0.5 to 3.996. Coproporphyrin levels ranged from 4.8 to 9r.z g per roe ml urine. The activity of ALA dehydratase was markedly decreased in all patients, ranging from 10 to 40:/o of normal, with a mean of 0.263 ,umoles of porphobilinogen synthe-
I
50 .I00 Pb in BlOOd pg/K)Oml
150
Pb in Urine
pg/lOOml
Fig. I. Correlation between x2.lues for lead in blood and ALA dehydratasc activity in red blood cells (REV), AL)\ dehydratasc actix-ity is expressed as ,~tmoles of prphobilinogcn synthcsizcd per ml of packed red blood cells per hour of incubation. +z 7 IL, Y : -0.75, p < -o.or. 1:jn o. 2. Correlation between x-alues for lead in urine and AL.4 cells (IWC). II =- 12, I, = -o.& p 7 0.2.
tlehytlrntase
activity
in ~-cd blood
# 0.5r .c 0.4: ;; 0.3$
: .
l.
20.2-
.
8
G
Ghi;hkk; ALA
in Urine
.
0.1
d
10
20
30
40
COPROPORPHYRIN
mg/‘iOOml
I-ix. 3, Correlation bctneen xxlues for ALA cells (RX). ~7 : IL, Y E ---0.76, fl < 0.01.
in urine and AL.4
0.6 0.5r
2
0 0.5-
x .
.c 0.4 _
.s $ 0.4
z s 0.3. 2 7II 20.2.
l. .
.
8 y 0.3,
.
l
.
60
dchydratasc
7oi%eo
flgf100ml
activit),
in rctl blood
tlehytlratasc
activity
l.
.
.
. .
. .
$0.2. 0
.
Q O.lr
in
‘: . .
D
‘.
&
50 in Urine
I’ig. 1, Correlation between x~~lues for coproporphyrin in urine and ;\LA IIT1 blood cells (KRC). ?1= 12, Y = -o.lg*, p : non-significant.
”
. .
. .
.
..
. .
SJ 0.1Q 2 RETI:“LOCYTES
Fig. 5. Correlation blood cells (RBC).
3
4
%
of
between values for reticulocytes in blood and AL.4 n = IL, Y = 0.05, p = non-significant.
Fig. 6. Correlation between the duration of exposure in red blood cells. n = 17, Y = -0.61, $J < 0.05. Clin. C/hiv~. Acta,
5 DURATION
19 (1968) 319-325
10 EXPOSURE
15 to Pb
dehydratase
to lead and the activity
of ALA
20 Years
activity
in red
dehydratasc
ALA
DEHYDRATASE
IN LEAD
POISONING
323
sized per ml of packed red blood cells per hour of incubation.
No correlation
was found
between the number of stippled cells and the other laboratory tests. Figs. I to 5 show the correlations between the activity of ALA dehydratase and the values for lead, ALA, coproporphyrin, or reticulocyte count. The decrease of ALA dehydratase activity in lead-poisoned erythrocytes correlated very closely ($J < 0.01) with the levels of lead in whole blood and ALA in urine, showing no clear correlation with tlie values for lead in urine, coproporphyrin in urine, and reticulocyte count in peripheral blood. Table II shows the activity of AL,4 dehydratase in erythrocytes obtained from patients with various kinds of neurological and hematological diseases. Marked decrease in ALA dehydratase activity was found only in the lead-poisoned erythroTABLE
II
Iliclg,zosis (X0. Of patlmts) Normal
A LA
dehvdvnfnsr
nctioit~“*
(IL)
Pb poisoning symptoms (+) (12) Symptoms (-~) (17) As poisoning (I) CO poiwning (I) Infectious polyneuritis (3)
0.
102+
O.‘)O
_I
0.263
I 0.081*
Cl..+01 1~ o.r81*
0.794 0.967 0.930,
0.902,
Diabetic neuropsthy (2) 0.889, 0.912 Subacute myelooptico0.989, 0.826 neuropathy (4) 0.698, 0.849 Acute intermittent porphyria (2) 0.998, x.08 Dejerinc-Sottas’s disease (I) 0.879 Multiple sclerosis (2) 0.798, 0.882 Amyotrophic
lateral
sclerosis(z)
Meningitis t,uberculosa \Vilson’s disease (I) Parkinsonian syndrome
(I) (2)
* Mean -1 S.1). ** Activity of _%LA dehvdratase packed red blood cells per hour
0.771, 0.912 0.768 0.776,
~1.901
0.698
is expressed of incubation.
0.894
Subacute combined dcgcneration (r)
0.u ‘J
Dystrophia
0.891, 0.709,
musculorum
ProB. (4) Polymyositis (I) Myotonia dystrophica(r) Brain tumor (2) Spinal tumor (I) Cervical spondylosis (L) iron deiiciency anemia (3) Hemolytic ar:cmia (2) htcgaloblastic anemia(r) Hypoplastic anemia (3) Sideroachrestic
anemia
(I) :\cutc myclocytic lcukcmia (7) Chronic myclocytic leukemia (1) as /~molcs porphobilinogen
0.990 1.120
0.05’
0.881
0.890.
1.Oj.j
0.751 0.698, I.Z~O, 0.889
0.890 1.309
1.360,
I..+O.j
0.685,
0.709 0.998
C.OOj,
0.599 I.055 0.780,
0.695
0.668,
0.881
synthesized
per ml of
cytes, though only a slight difference in the activity was found between patients with symptoms of lead poisoning and workers exposed to lead without such symptoms. Fig. 6 shows the relationship between the decrease of ALA dehydratase activity and the duration of exposure to lead. Decrease of the enzyme activity was already found in workers after 3.2 years of exposure, and a close correlation was demonstrated between the length of time and the decrease of enzyme activity. Although isolation and discontinuation of the exposure to lead for a month improved ALA dehydratase activity but slightly, a statistically significant (fi < 0.05) recovery in the enzyme activity was found in workers who had received injections of reduced glutathione, as compared with those without injections (Table III). Clin. Chim. Acta,
19 (1968)
3x9-325
_ I 2
I
3
\
4 5 Mean
6 87
-‘:
Isolation
from exposure
to lead alone
S.D.
0.32
0.25
0.29
0.04
0.22
0.32
0.10
0.26
0.29
0.o.j
0.21
0.22
0.01
0.26
\
9
0.3.5
Isolation
from exposure to lead plus of reduced injections glutathione**
_‘~ 0.05
0.24
0.20 o.24
IO
0.34 0.29
Mean & S.D.
0.26 ~~ o.oj
-0.0.3
o.oq 0.39
0.37 0.30 0.46 0.37
:y- 0.0-l
0.l.j
0.06j 0.1 0.12
0.08 0.12
I 0.o.J I’. -: 0.05
* Exprcsscd as ,umoles of porphobilinogen synthesized per ml of packed red blood cells per hour of incubation. ** Reduced glutathionc was injected intramuscularly in doses of zoo mg/day for 30 days. DISCUSSIOIi
Several authors reported that increased urinary excretion of ALA appears to be closely correlated with lead poisoningr*rr9r2. Furthermore, Holmqvist13, in an investigation on 149 workers, found a good correlation between lead in the blood and exposure to lead. On the other hand, De Kretser and Waldron14 found only a poor correlation between ALA and the urinary lead output in an investigation on roe lead workers, whereas Haeger-Aronsenl found a good correlation. The present study showed that the activity of ALA dehydratase was markedly decreased in almost all the lead-poisoned erythrocytes, and that the decrease of the enzyme activity was closely correlated with the increased blood level of lead and urinary level of ALA, and with the duration of the exposure to lead. These results indicate that the measurement of ALA dehydratase activity in the erythrocytes offers a good and simple method of evaluation of lead poisoning, in view of the difficulties involved in the determination of ALA. Lead in the blood is said to be rapidly incorporated into red blood cells and probably affects ALA dehydratase 1,)~ directly inactivating the sulfhydryl groups necessary for its activitys. Considering the facts that the peripheral mature erythrocytes do not actually synthesize ALA and that the increased ALA probably originates from immature erythroid cells in the bone marrow, a good correlation between urinary ALA and erythrocyte ALA dehydratase activity seems to indicate the possible inhibition of ALA dehydratase of immature erythroid cells in the bone marrow along with that of mature erythrocytes in the peripheral blood. As can be seen in Table II, neurological and hematological disorders resembling lead poisoning could be differentiated from lead poisoning by measuring ALA dehydratase activity in erythrocytes, although this enzyme activity might not discriminate between clinically obvious cases of lead poisoning and latent ones. Lichtman and Feldman5 demonstrated the reversal of the ALA dehydratase deficit in C&n. Chim.
Acta,
19 (1968)
319-325
ALA DEHYDRATASE
IN LEAD POISONING
325
the lead-poisoned erythrocytes by preactivating the incubation mixture with glutathione. In the present study, in viva administration of reduced glutathione in workers with lead poisoning improved the decreased ALA dehydratase activity to some extent. For the recovery of ALA dehydratase activity, reduced glutathione seems to be effective in vivo as well as in vitro. ACKNOWLEDGEMENT
We thank
Drs. H. Katsunuma
and T. Suzuki
for their kind helps.
REFERENCES I B. HAEGER-ARONSEN, Stand. J.Clin. Lab. Iwest., Suppl., 12 (1960) 47. AND J. J. CHISOLM, J. Pediat, 56(1960) 759. 3 K. D. GIBSON, A. NEUBERGER AND J. J. SCOTT, B&hem. J.. 61 (1955) 618. 4 E. I. B. DRESEL AND J. E. FALK, B&hem. J., 63 (1956) 80. 5 H. C. LICHTMAN AND F. FELDMAN, J. Clin.Invesl., 42 (1963) 830. 6 A. GOLDBERG, J. A. SMITH AND A. C. LOCHHEAD, Brit. Med.J., i(Ig63) 1270. 7 D. ~M~UZERALL AND S. GRANICK, J. Biol.Chem., zig (1956) 435. 8 C. RIMINGTON, Assoc. Clin.Pathologists, Broodsheet N. S., No. 21. Nov. (1958). 9 A. A. MONCRIEFF, 0. P. KOUMIDES, B. E.CLAYTON,A. D. PATRICK,A.G.C. RENWICK G. E. ROBERTS, Arch. Diseases Childhood, 39 (1964) I. IO S. GRANICK AND D. MAUZERALL, J.Biol. Chem., 232 (1958) Irrg. II R. C. GRICCS AND J. W. HARRIS, Clin.Res., 2 (1958) 188. 12 K. CRAMI?R AND S. SELANDER, Brit.J. Ind.Med., 22 (1965) 311. 13 I. HOLMQVIST, Schri_ften Ges. Deut. Metallhiitten und Bergleute, 7 (1960) 115. 14 A. J. DE KRETSER AND H. A. WALDRON,BY~~. J. Ind. Med., zo(rg63) 35. 2 P. E. BENSON
C&z. Chim. Acta,
19 (1968)
AND
319-325