Clinical Science (1983) 64,187-191
187
Changes in haem synthesis associated with occupational
exposure to organic and inorganic sulphides
R. T E N H U N E N * , H . S A V O L A I N E N T A N D P . J A P P I N E N S
*Department of Clinical Chemistry, University of Helsinki, Helsinki, ?Department of Industrial Hygiene and Toxicology,
Institute of Occupational Health, Helsinki, and $Medical Station, Enso-Gutzeit Oy, Imatra. Finland
(Received I0 May I982/4 August 1982; accepted I0 September 1982)
Summary
1. Analysis of reticulocytes for Gaminolaevulinic acid synthase (AmLev synthase, EC
2.3.1.37) and haem synthase (EC 4.99.1.1)
activity in 17 workers in pulp production with
low-level hydrogen sulphide and methylmercaptan exposure showed decreased activities
in eight and six cases respectively.
2. Erythrocyte protoporphyrin concentration
was below the control range in seven cases.
3. Low AmLev synthase and haem synthase
activities were found in one patient with hydrogen
sulphide intoxication 1 week after the event. The
activities had returned to the control levels 2
months later, though erythrocyte protoporphyrin
remained abnormally low.
4. In vitro, hydrogen sulphide inhibited haem
synthase with an apparent Kl of 3.4 mmol/l.
Sulphide anion, on the other hand, inhibited
AmLev synthase activity 85% at 10 mmol/l
concentration. Thiosulphate anion inhibited AmLev synthase activity 18% (Kl 27 mmol/l) and
haem synthase activity 43% at 10 mmol/l
concentration. Selenite inhibited AmLev synthase (K,5 - 1 mmol/l) and haem synthase (K,9.0
mmol/l).
5. The assay of AmLev synthase and haem
synthase could be a valuable addition to the
assessment of workers’ health in industries
generating hydrogen sulphide or/and methylmercaptan, although the mechanism of the toxic
effect remains speculative.
Key words: Gaminolaevulinic acid dehydratase,
Gaminolaevulinic acid synthase, coproporCorrespondence: Dr Raimo Tenhunen, Department
of Clinical Chemistry, University of Helsinki, Meilahti
Hospital, SF-00290 Helsinki 29, Finland.
0143-5221/83/020187-05$2.00
phyrinogen oxidase, haem synthase, haem synthesis, sulphides, uroporphyrinogen decarboxylase, uroporphyrinogen synthase.
Abbreviations: AmLev, 8-aminolaevulinic acid;
AmLev dehydratase, Gaminolaevuliiic acid
dehydratase; AmLev synthase, S-aminolaevulinic acid synthase.
Introduction
Acute hydrogen sulphide intoxication is an
acknowledged occupational hazard in, for example, the petroleum industry and in the production
of cellulose by the chemical process ill. Hydrogen sulphide and simple organic sulphides are
known inhibitors of cellular respiration at minute
concentrations [21, which explains the lifethreatening nature of acute intoxication [31.
Clinical experience indicates that undesirable
effects on health may also be caused by low-level
or repeated exposure to hydrogen sulphide,
although the pathogenetic mechanisms are virtually unknown [41. We have demonstrated
previously that the inhibition of cerebral cytochrome oxidase in four sequential subclinical
hydrogen sulphide intoxication episodes may be
cumulative in mice [51. This has led us to
investigate the synthesis of haem, a prosthetic
group of haemoglobin and most cytochromes, in
workers with known exposure to hydrogen
sulphide and methylmercaptan.
The biosynthetic pathway of haem is virtudly
identical in the bone marrow and in liver 161, and
the regulatory enzyme activities in man have been
elucidated in porphyric diseases. GAminolaevulinic acid synthase (AmLev synthase, EC
2.3.1.37) is the rate-limiting step, with haem-
Q 1983 The Biochemical Society and the Medical Research Society
188
R . Tenhunen,H . Saoolainen and P. Jappinen
induced feed-back inhibition and repression by an
unknown mechanism 17-91.
Materials and methods
Venous blood was collected, with heparin as an
anticoagulant, and centrifuged at 1000 g for 10
min at +4OC. Plasma and buffy coat were
discharged. The remaining red cells were
resuspended in cold 0.9% NaCl solution to
achieve a dilution of (2-3) x 1 0 I 2 red blood cells
per litre. The number of the red cells and
percentage of the reticulocytes were counted by
routine methods [lo]. To the final red-cell
suspension, 50 pl of Tween-2O/ml of suspension
was added and the cells were sonicated for 2 min
in an ice bath with 50% duty cycle, setting 4, by a
pulsed sonifier (Sonifier, model B-30; Branson
Sonic Power Co., Danbury, CT, U.S.A.). The
sonicates were stored in liquid nitrogen until used
for incubation. No loss of enzymic activities were
detected during storage.
The activity of AmLev synthase was measured
by the method of Ebert et al. I l l ] with the
following modification: maximal rates of 6aminolaevulinic acid (AmLev) formation required addition of exogenous succinic thiokinase
(0.5 unit), CoA (106 mmol/l) and guanosine
triphosphate (96 mmol/l) for generation of
succinyl-CoA. Furthermore, the final incubation
mixture contained separately tested optimal
concentrations of glycine (100 mmol/l). Tris/
HCI, pH 7.4 (40 mmol/l), pyridoxal 5-phosphate
(0.2 mmol/l), EDTA (0.5mmol/l) and 1 pCi of
[2,3-14Clsuccinicacid and 0.8 ml of the red-cell
sonicate was added to incubation mixture with a
final volume of 2 ml. The incubation time was 40
min at 37OC. The enzyme activity was expressed
as pmol of labelled Gaminolaevulinic acid formed
in 1 h per lo6 reticulocytes. The recovery of the
added AmLev was between 80 and 90%. The
radioactivity of labelled [14C)AmLev was
measured by a Wallac 8 1 000 liquid-scintillation
counter.
12,3-14C1Succinic acid (sp. radioactivity 50
mCi/mmol) was purchased from The Radiochemical Centre, Amersham, Bucks., U.K.
Glycine, succinic thiokinase, pyridoxal 5phosphate, CoA, GTP and Tris/HCI were
purchased from Sigma Chemical Co., St. Louis,
MO, U.S.A. 14-14ClAmLev hydrochloride (sp.
radioactivity 25 mCi/mmol) was obtained from
New England Nuclear Corp., Boston, MA,
U.S.A.
The activity of Gaminolaevulinic acid dehydratase (AmLev dehydratase, EC 4.2.1.24) was
measured by following the instructions of Euro-
pean standardized method [12]. AmLev used in
the assay was purchased from Sigma. The
activity of uroporphyrinogen I synthase (EC
4.3.1.8) was measured by the method of Strand et
al. [ 131, as modified by Mustajoki [ 141. Porphobilinogen used in this assay was obtained from
Sigma.
The activity of uroporphyrinogen decarboxylase (EC 4.1.1.37) was measured by modifying the method of Romeo & Levin 1151. The
incubation system included 0.1 ml of red-cell
sonicate, potassium phosphate buffer (0.067
mmol/l), pH 7.0, glutathione (2 mmol/l) and
uroporphyrinogen I (14 mmol/l). The incubation time was 2 h at 37OC. Uroporphyrin I
used in this assay was obtained from Porphyrin
Products, Logan, UT, U.S.A. The activity of
coproporphyrinogen oxidase (EC 1.3.3.3) was
measured by a high-performance liquidchromatographic method of Salmi & Tenhunen
[ 161. The incubation time was 2 h at 37OC.
A slightly modified method by Bonkowsky et
al. 1 171 was used to measure the activity of haem
synthase (EC 4.99.1.1). Red-cell sonicate (0.5
ml) was added to incubation mixture, which
otherwise contained the same amounts of the
reagents as described by Bonkowsky et al. [ 171,
except that the concentration of protoporphyrin
was 180 mmol/l. The incubation time was 45 min
at 37OC. The final iron working solution contained 2 pCi of 59Feand Fez+ (500 mmol/l) per
50 pl. The radioactivity of the 59Fe-labelledhaem
was measured with a Wallac 80000 gamma
sample counter. Enzyme activity was expressed
as pmol of 59Fe-labelled haem formed per lo6
reticulocytes in 1 h.
Protoporphyrin dimethyl ester was purchased
from Sigma. Ferrous sulphate (0.2 mCi of
sgFe/ml and 33 mg of ascorbic acid/ml, pH 1-2)
was purchased from Isocommerz, Dresden,
German Democratic Republic.
Red-cell protoporphyrin concentrations were
determined by a high-performance liquidchromatographic method of Salmi & Tenhunen
1161.
Enzyme measurements in inhibition studies by
sulphide, sulphite, thiosulphate and selenite were
carried out as in the Materials and methods
section, except that 7000 g rat liver homogenate
was used as the enzyme source.
Protein was determined by the method of
Lowry et al. 1181. A cross-sectional hygiene
study measuring hydrogen sulphide, methylmercaptan and dimethylsulphide concentrations
in the air of working place was carried out. This
was completed with clinical surveys about possible subjective symptoms in the exposed wor-
189
Sulphide eflects on haem synthesis
kers. Fifty age-matched clerical staff served as
controls.
drawn 1 week after the event. AmLev synthase
and haem synthase activities were low but
returned to normal in 2 months (Table 2).
Erythrocyte protoporphyrin was low even at that
time point.
In a cross-sectional hygienic survey, the
concentration of hydrogen sulphide ranged from
0.05 to 5.2 p.p.m., calculated as a time-weighted
average over 8 h. The methylmercaptan concentration varied from 0.07 to 2.0 p.p.m., and
0.03 to 3.2 p.p.m. dimethylsulphide was also
found. In a clinical survey of subjective
symptoms by predistributed questionnaires, no
complaint appeared more often than in the
control group. However, the average annual
number of days on sick leave tended to be higher
(12.9 as against 7.1).
.
Hydrogen sulphide anion failed to inhibit
AmLev synthase activity in vitro, while it inhibited
Results
AmLev synthase activity was below the control
range in eight out of seventeen workers examined,
haem synthase in six of the seventeen and AmLev
dehydratase in two. The erythrocyte protoporphyrin was below the control range in seven
cases, notably in those with simultaneous depression of AmLev synthase and haem synthase
activity. Uroporphyrinogen I synthase and uroporphyrinogen decarboxylase activities were
within the control ranges, as were the erythrocyte
coproporphyrin concentrations (Table 1).
In the course of our study, one acute hydrogen
sulphide intoxication occurred. The patient was
initially unconscious, and a blood sample was
TABLE1. Activities of haem-synthesizing enzymes in sulphide and rnethylmercaptan-exposedmale workers
The activity of Gaminolaevulinic acid synthase (AmLev synthase) is indicated as pmol of AmLev h-' (10' reticu1ocytes)-', of
6-aminolaevulinic acid dehydratase (AmLev dehydratase) as pmol of porphobilinogen h-' ( 10' erythrocytes)-', of
uroporphyrinogen I synthase as pmol of uroporphyrinogen h-' (mg of protein)-', of uroporphyrinogen decarboxylase as
pmol of coproporphyrin h-' (me of protein)-', of coproporphyrinogen oxidase as pmol of protoporphyrin h-' (10'
reticu1ocytes)-' and that of haem synthase as pmol of haem-' ( 10' reticu1ocytes)-I. The erythrocyte porphyrin concentrations
are given as nmol/l of erythrocytes.
Case
1
2
3
4
5
6
7
8
9
10
II
I2
13
14
15
Age
CoproHaem
Years AmLev
AmLev Uroporphyrinogen Uroporphyrinogen
in the synthase dehydratase
I synthase
decarboxylase porphyrinogen synthase
work
oxidase
42
57
43
55
45
53
56
46
46
52
53
51
32
59
42
10
20
18
40
23
30
40
20
21
18
20
18
14
20
17
Reference values
101
206
109
142
4.6
3.5
28.6
27.2
7.4
7.5
3.4
19.7
6.7
11.2
11.8
7.0
1.9
3.2
29.8
99-164
2.2-6.5
6.1-21.9
117
108
133
131
138
117
151
138
144
137
134
121
-
103
88
84
68
84
95
77
76
84
96
71
92
88
79
80
0.16-0.84
227-462
56-106
0.09
ProtoCoproporphyrin porphyrin
5.5
10.5
34.8
4.0
4. I
6.2
2.4
4.9
3.0
7,4
3.7
3.4
3.0
3.0
3.4
308
248
303
238
227
24 1
277
434
152
296
322
204
252
319
230
0.18
0.46
0.26
0.22
0.15
0.02
0.04
0.01
0.08
0.01
0.03
0.17
Erythrocyte
-
3.8
9.3
-
-
-
195
208
101
291
375
258
258
441
324
158
123
6.7
3.5
10.0
4.1
4.3
3.0
4.5
5.1
6.2
13.0
3.3
220-934
2.2-30
TABLE2. The enzyme activities of haem synthesis in blood of a person with an acute hydrogen sulphide intoxication I week
and 2 months d e r the incident
The units of enzyme activities as well as the reference values are given in Table 1. All enzyme determinations were performed
in triplicate.
Time since AmLev
AmLev
Uroporphyrinogen Uroporphyrinogen
CoproHaem
Erythrocyte
intoxication synthase dehydratase
I synthase
decarboxylase porphyrinogen synthase
oxidase
Protoporphyrin Coproporphyrin
I Week
2Months
0.06
0.29
404
340
76.6
81.0
143
118
2.4
6.2
3.7
7.4
39
65
4.4
6.2
R . Tenhunen,H . Savolainen and P . Jappinen
190
TABLE3. Inhibitory effects of selenite. sulphide, sulphite and thiosulphate anions on AmLw
synthase and haem synthase in vitro
AmLev synthase and haem synthase activities were determined as described in the Materials
and methods section using rat liver mitochondria as the enzyme source. All measurements
were performed in triplicate. The enzyme inhibition (%), range of results (in parentheses) and
K,(mmol/l) are given. Abbreviation used: N.D., not determined.
Inhibitor
AmLev synthase
Concentration
Haem synthase
(mmolll)
% Inhibited
HS -
HS0,-
10
1
0.1
10
I
0.I
HS,O,-
10
1
HSe0,-
10
1
0.I
0.1
0
4
%Inhibited
Kl
N.D.
100(1OO-100)
3.4
0
0
85 (83-88)
0
0
18 (13-22)
8 (7-9)
0
78 (73-81)
36 (34-39)
6 (3-10)
the haem synthase with an apparent K, of 3.4
mmol/l. Sulphite anion inhibited AmLev synthase
activity 85% at 10 mmol/l concentration ( K , not
determined), while no inhibition was detected in
the haem synthase activity in vitro (Table 3).
Thiosulphate anion inhibited AmLev synthase
18% ( K i 27 mmol/lj and haem synthase 43% at
10 mmol/l concentration. Selenite inhibited AmLev synthase (K,5.1 mmol/l) and haem synthase
( K , 9.0 mmol/l) in vitro (Table 3).
Discussion
None of our cases had clinical anaemia, and
sternal-marrow smears made on five cases had a
normal cytological picture, although the yield of
marrow was scanty. This supports the idea that
extra-marrow haem synthesis may compensate
for the inhibition of the erythroid pathway [191.
The nature of the inhibition in the erythroid cell
line is not clear. Inhaled hydrogen sulphide is
known in mice to be metabolized to non-volatile
products [20].This might also be the case in
humans. Sulphide anion is oxidized in liver and in
kidneys to sulphite and thiosulphate 121, 221,
which undergo further oxidation to sulphate [231.
In our in vitro experiments, sulphide anion only
inhibited haem synthase, while sulphite inhibited
the AmLev synthase and thiosulphate inhibited
haem synthase and AmLev synthase activities. It
is interesting to note that prolonged hydrogen
sulphide vapour exposure has been reported to
cause reticulocytosis and increased red-cell corpuscular volume in 90 days in experimental
animals [241, and when sulphite was administered to rats (50 mmol/kg body wt. for 8 weeks,
daily) macrocytic anaemia occurred [231.
N.D.
27
5.1
62 (60-63)
0
0
0
0
43 (38-46)
0
0
53 (51-57)
23 (21-26)
16 (15-16)
N.D.
N.D.
9.0
Our in vitro data indicate that these findings
may be due to direct effects on the haemsynthesizing enzymes. Because of electronic
similarity with the sulphite anion, we also tested
selenite, which was an even more potent inhibitor
than sulphite. This might be an important
harmful effect of selenite toxicity, especially at
low doses, when acutely toxic effects are avoided.
To our knowledge, this is the first report to
describe cumulative biochemical effects in human
haem synthesis caused by low-level hydrogen
sulphide and methylmercaptan exposure.
Although tedious, the analysis of AmLev synthase and haem synthase activities could give
valuable information on the health of selected
worker groups, for example in the oil and pulp
industries. However, it is not known whether the
inhibition is caused by the low prevailing levels or
by cumulative effects of occasional peak concentrations.
Acknowledgments
We thank the participating workers and the staff
of the Medical Station of Enso-Gutzeit Oy for
their co-operation. The skilful technical
assistance of Mrs Aila Akerlund, Mrs Marita
Junna and Mrs Anja Pakkanen is gratefully
acknowledged. This work was supported by a
grant from the Sigrid Jusklius Foundation.
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