British Journal of Anaesthesia 1990; 64: 214-223
DIMETHYLTHIOUREA, A HYDROXYL RADICAL
SCAVENGER, IMPEDES THE INACTIVATION OF
METHIONINE SYNTHASE BY NITROUS OXIDE IN MICEf
D. D. KOBLIN AND B. W. TOMERSON
SUMMARY
Dimethylthiourea (DMTU), a potent scavenger of
hydroxyl radicals, was studied to see if it attenuated the inactivation of methionine synthase
produced by nitrous oxide in mice. Mice were
given i.p. injections of DMTU 0.5-4.0mg g-1
or saline and, 1 h after injection, were exposed
to 66% nitrous oxide in oxygen for periods of
0.5-8 h. At given times after nitrous oxide
exposure, higher methionine synthase activities
were found in the livers, kidneys and brains of
mice injected with DMTU than in the salineinjected animals. These higher methionine synthase activities in the DMTU-treated animals
represented a delay in the enzyme inactivation
produced by nitrous oxide, as the difference in
activities between the DMTU-injected and
saline-injected mice decreased with increasing
duration of exposure to nitrous oxide. Greater
differences in methionine synthase activities
between the DMTU- and saline-injected animals
were observed with increasing doses of DMTU.
The rate of enzyme inactivation following exposure to nitrous oxide was greater in liver and
least in brain, and the difference in activities
between the two groups varied with the organ
examined. DMTU exhibited its greatest effect in
the kidney, where methionine synthase activities
were nearly doubled in the DMTU 2.0 mg g~1 injected compared with the saline-injected mice
after 1-h exposure to 66% nitrous oxide. Following a marked inactivation of methionine
synthase by exposing mice to 66% nitrous oxide
for 4 h, injection of DMTU 2.0 mg g~' at the end
of exposure to nitrous oxide did not enhance, but
impaired, the recovery of enzyme activity. The
findings are consistent with the hypothesis that
nitrous oxide combines with the vitamin B12
molecule of methionine synthase to form a
hydroxyl radical that reacts with and inactivates
the enzyme, and that DMTU slows this inactivation by scavenging hydroxyl radicals.
KEY WORDS
Anaesthetics gaseous: nitrous oxide. Enzymes: methionine
synthase. Dimethylthiourea. Vitamin B12.
Nitrous oxide inactivates the vitamin B12dependent enzyme methionine synthase (EC
2.1.1.13) in animals [1-3] and humans [4, 5]. Following acute exposure of rodents to nitrous oxide
and inactivation of methionine synthase, there is
slow recovery of enzyme activity in room air and
4 days or longer may be required for complete
recovery [6-10]. Although it is uncertain why
such a prolonged period is needed, it is likely that
reaction with nitrous oxide causes damage both to
the vitamin B12 molecule and to the enzyme, and
that new apoenzyme needs to be synthesized [11].
Recent biochemical studies with the isolated
and purified enzyme suggest a possible mechanism by which the nitrous oxide-induced oxidation of the vitamin B12 molecule might lead to
enzyme damage [12, 13]. It was proposed that
nitrous oxide acts by one-electron oxidation of the
cob(I)alamin form of the enzyme which is generated transiently during turnover, with the
formation of cob(II)alamin, nitrogen and hydroxyl radical, according to the following scheme:
DONALD D. KOBLIN*, PH.D., M.D. ; BARBARA W. TOMERSON,
M.S.; Departments of Anesthesia, Veterans Administration
Medical Center, San Francisco, CA 94121, and University of
California, San Francisco, CA 94143, U.S.A. Accepted for
Publication: July 4, 1989.
*Address for correspondence: Anesthesiology Service
(129), Veterans Administration Medical Center, 4150 Clement
Street, San Francisco, CA 94121, U.S.A.
fA preliminary report of these results was published in
Anesthesiology 1988; 69: A434.
DIMETHYLTHIOUREA, METHIONINE SYNTHASE AND N2O
+
cob(I)alamin + N2O + H
^•cob(II)alamin + N 2 + "OH (1)
It was speculated that the hydroxyl radical, which
is extremely reactive [14], might attack amino acid
residues near the active site of the enzyme and
lead to irreversible inactivation of methionine
synthase [12, 13].
If the above supposition is correct, a compound
that scavenges free hydroxyl radicals and is able to
penetrate into the cytoplasm where methionine
synthase is located, might protect the enzyme
from inactivation by nitrous oxide. 1,3-Dimethylthiourea (DMTU) [CH3NHCSNHCH3] is an
effective scavenger of hydroxyl radicals and is
highly permeable to cell membranes [15]. The
administration of DMTU ameliorates cellular
damage associated with the production of hydroxyl radicals in several tissues. For example, at
doses of 0.5-1.0 g kg"1, DMTU prevents granulocyte-mediated oxidant lung injury in rats [15,
16] and re-expansion pulmonary oedema in
rabbits [17], and attenuates endotoxin-induced
respiratory failure in pigs [18]. DMTU lessens
reperfusion injury after short episodes of renal
ischaemia [19] and protects against gentamicininduced acute renal failure [20] in the rat. DMTU
also protects the rat heart against reperfusion
injury after an episode of ischaemia [21] and
improves contractile function after regional ischaemia of the dog myocardium [22]. In the
present investigation, we tested the ability of
DMTU to impede the inactivation of methionine
synthase in the livers, kidneys and brains of mice
exposed to nitrous oxide.
METHODS
Animals
Adult male ICR mice (Simonsen Labs, Gilroy,
CA), initially weighing about 30 g, were provided
Purina Rodent Laboratory Chow (Diet No. 5001,
Purina Mills, Inc.) and tap water ad libitum except
during the periods of exposure to nitrous oxide.
These studies were approved by the Animal
Studies Subcommittee at the Veterans Administration Medical Center, San Francisco.
215
Inc., Portland, OR), prepared as a solution of
DMTU 100 mg/ml isotonic saline (0.9% NaCl),
at a dose of 2.0 mg/g body weight. Alternatively,
mice received i.p. injections with an equivalent
volume of saline. One hour after the injections,
four DMTU-injected mice and four salineinjected mice were inserted into individual wire
mesh cages and placed in a 20-litre stainless steel
chamber [23]. A mixture of nitrous oxide 5 litre
min"1 and oxygen 2 litre min"1 was administered
via an Ohio anaesthesia machine for 10 min, and
the gas flows were then decreased to 2 litre min"1
and 1 litre min"1, respectively, for the duration of
the exposure. Exposures were carried out for
30 min and 2, 4 and 8 h. The stainless steel
chamber was equipped with a fan to circulate gases
through a soda-lime container to remove carbon
dioxide [23, 24]. The chamber temperature remained near 23 °C, but varied between 20 and
25.8 °C. Gas samples were removed from the
chamber during the exposures, and oxygen concentrations were measured with a Beckman Model
E-2 oxygen analyser. Oxygen concentrations
varied between 32.1 and 35.1%. Gas analyses
using a SARA mass spectrometer confirmed
nitrous oxide concentrations to be approximately
66 % and carbon dioxide concentrations to be less
than 1 %. Exposures were carried out between
07:00 and 17:00. At the end of the period of
exposure to nitrous oxide, the chamber was
flushed with 100% oxygen approximately 10 litre
min"1 for 2 min. The animals were killed immediately with 100% carbon dioxide, and livers,
kidneys and whole brains were isolated from the
animals and stored at — 20 to — 30 °C until
analysed for methionine synthase activity. Organs
were isolated also from a control group (n = 8) of
mice not injected with DMTU or exposed to
nitrous oxide.
In the following sets of experiments the
methods for DMTU injections, conditions of
exposure to nitrous oxide, analysis of gas samples
and tissue removal procedures were the same as
described above.
(2) Dose-dependence of DMTU. Mice received
i.p. injections of DMTU 0.5,1.0, 2.0 or 4.0 mg/g
body weight or an equal volume of saline. One
hour after the injections, four DMTU-injected
DMTU treatments and exposures to nitrous oxide and four saline-injected mice were exposed to
66% nitrous oxide in oxygen for 1 h. These
Four sets of experiments were performed:
(1) Increasing duration of exposure to nitrous animals were killed immediately after exposure to
oxide. Mice were given intraperitoneal (i.p.) nitrous oxide. A control group (n = 4) was not
injections of DMTU (American Tokyo Kasei, injected or exposed to nitrous oxide. A DMTU
216
control group (n = 4) was given DMTU 2.0 mg g 1
but not exposed to nitrous oxide. These animals
were killed 2 h after the DMTU injections.
(3) Duration of protection by DMTU. Mice
were injected with DMTU 2 mg/g body weight
or an equal volume of saline. At 15 min or 1, 4 or
16 h after the DMTU injections, four DMTUinjected and four saline-injected mice were exposed for 1 h to 66 % nitrous oxide in oxygen.
The mice were killed immediately following
exposure to nitrous oxide. A control group of mice
(M = 4) was not injected or exposed to the gas.
DMTU controls (n = 4) were injected with
DMTU 2 mg g"1, but not exposed to nitrous
oxide and killed within 10 min of the DMTU
injections. Before organ removal, blood was taken
from the inferior vena cava using a 25-gauge
needle,, the serum separated after centrifugation at
1000 £ and frozen until analysed for DMTU.
(4) Effect of DMTU on recovery of methionine
synthase activity. Groups of eight mice were
exposed for 4 h to 66 % nitrous oxide in oxygen.
Immediately after exposure to nitrous oxide, four
mice were injected with DMTU 2 mg g"1 and
four mice were injected with an equal volume of
saline. Each group of eight mice treated in this
manner was killed 6 h, 16 h, 2 days or 4 days after
injection of DMTU. A control group (« = 4) that
was not injected and not exposed to nitrous oxide
was also killed. Another group (n = 4) was not
injected, but was exposed to 66% nitrous oxide
for 4 h and killed immediately after the exposure.
BRITISH JOURNAL OF ANAESTHESIA
conditions of the Hewlett-Packard 5830A gas
chromatograph used for analysis included a
nitrogen flow through the column of 30 ml min"1,
injector and flame ionization detector temperatures of 260 °C, and the oven temperature
initially set at 120 °C for 2 min and then programmed to increase to 155 °C at 30 °C min"1 and
remain at 155 °C for 6 min. The DMTU peak
appeared at 0.89 min and the diethyl sulphone
peak at 7.67 min. The coefficient of variation for
the assay was 6.0%.
Statistical analysis
Statistical comparisons between the DMTUand saline-injected groups were performed with
an unpaired t test. Of the 156 mice examined in
these experiments, one mouse died when injected
with DMTU following exposure to nitrous oxide.
This was the only animal eliminated from statistical analysis. All values are expressed as the
mean (SD).
RESULTS
In the first series of experiments, methionine
synthase activities were determined in mice
injected with saline or with a single dose of
DMTU 2.0 mgg" 1 i.p. and exposed to 66%
nitrous oxide for various periods. Methionine
synthase activities decreased progressively in liver
(fig. 1 A), kidney (fig. 1 B) and brain (fig. 1 c) with
increasing duration of exposure to nitrous oxide.
The rate of enzyme inactivation, for either
DMTU-injected or saline-injected mice, was
Assays
greatest in liver and least in brain. In liver (fig.
Methionine synthase activity was determined as 1 A), more than 50 % of the enzyme was inactivated
described previously [2]. Activity was expressed after 30 min of exposure to nitrous oxide, whereas
as nanomoles of methionine produced per hour in brain (fig. 1 c), less than 50 % of the enzyme
was inactivated after 2 h of exposure to nitrous
per gram of original tissue.
DMTU concentrations in serum were quan- oxide. DMTU impeded the inactivation of methtitated by a modified gas chromatographic pro- ionine synthase by nitrous oxide in all three
cedure [25]. Ten microlitre of a 3.5-mg ml"1 organs. Compared with the saline-injected anisolution of diethyl sulphone (internal standard) mals, significant increases in methionine synthase
was added to 200 ul of serum isolated from a activity were found in DMTU-treated animals.
DMTU-treated mouse. The sample was mixed The higher methionine synthase activities in the
after addition of 40 ul of 1.8-mol litre"1 perchloric DMTU-treated animals represented a delay in
acid, allowed to incubate at 0 °C for 10 min, and the enzyme inactivation produced by nitrous
centrifuged. One hundred microlitre of the super- oxide, as the difference in enzyme activities
natant was removed, 20 ul of 2-mol litre"1 between the DMTU-injected and saline-injected
K 2 HPO 4 added, the solution mixed, incubated at mice tended to disappear with increasing duration
0 °C for 5 min, and centrifuged. One microlitre of of exposure to nitrous oxide. In liver (fig. 1 A),
the supernatant was injected onto a 60-cm long, 2- significant increases in methionine synthase
mm i.d. glass column packed with 20 % Carbowax activity occurred in the DMTU-treated mice for
20 M on Supelcoport 80/20 mesh. Operating as long as 2 h after the start of exposure, but
217
DIMETHYLTHIOUREA, METHIONINE SYNTHASE AND N2O
r
200-1
^T 3 0 0 n
o ~ o Saline
• — • DMTU 2 mg g"1
- 200-
O—O Saline
•—•DMTU2mgg -1
100-
'H--^:
r
o
I
8
2
4
6
Exposure to 66/i N2<D(h)
Exposure to 66% N20(h)
_" 200o--o Saline
•—• DMTU 2 mg g"1
100-
o-i
2
4
6
I
8
Exposure to 66% N2O (h)
disappeared by 4 h. In kidney (fig. 1 B), significant
increases in enzyme activity were detected at 0.5,
2 and 4 h after the start of exposure in the
DMTU-injected animals, but not at 8 h. In brain
(fig. lc), significant increases in methionine
synthase activity were seen in the DMTU-treated
mice even 8 h after starting exposure to nitrous
oxide. DMTU exhibited its greatest effect in
kidney after a 30-min exposure to nitrous oxide;
at this point methionine synthase activities (expressed as nmoles of methionine produced per
hour per gram of kidney (SD)) were 208 (21.8) in
the DMTU-injected and 130 (12.7) in the salineinjected mice (fig. 1 B).
In the second set of studies, the dose-dependence of DMTU was examined in animals
exposed to 66% nitrous oxide for 1 h. Hepatic
methionine synthase activities were greater in the
DMTU-injected compared with the saline-injected mice at each of the DMTU doses. However,
FIG. 1. Methionine synthase activities in livers (A), kidneys
(B) and brains (c) of mice injected with DMTU 2 mg g"1 or
saline 1 h before exposure to 66 % nitrous oxide in oxygen for
periods of 0.5, 2, 4 or 8 h. The point at time 0 represents
animals that were not injected and not exposed to nitrous
oxide. Each point was determined from four separate animals.
Errors indicate SD; if no error bars are shown, errors are
within the size of the symbols. Activities were compared
between the DMTU- and saline-injected mice at each time
point (except time 0), and significance was calculated using an
unpaired t test: *P < 0.05; **P < 0.01; ***P < 0.001.
only the highest dose of DMTU tested (4.0 mg
g"1) produced a significant increase in this series
of experiments (fig. 2 A). In kidney (fig. 2 B) and
brain (fig. 2 c), methionine synthase activities
were significantly higher even at the lowest dose
of DMTU used (0.5 mgg-1)- With increasing
doses of DMTU, there was a greater separation in
methionine synthase activities between the salineinjected and DMTU-treated groups. Methionine
synthase activities were also measured in control
mice that did not receive an injection and were not
exposed to nitrous oxide, and in mice that were
injected with DMTU 2.0 mg g'1 and not exposed
to nitrous oxide. In these mice that were not
exposed to nitrous oxide, DMTU had no significant effect on activities in liver, kidney or brain
(fig. 2 A, B, c).
In a third series of experiments, mice were
given i.p. injections of DMTU 2 mg g"1 or saline
and allowed to remain in room air for various
BRITISH JOURNAL OF ANAESTHESIA
218
•7
O)
200-,
\
300o
c
D Saline
100a***
1.0
2.0
4.0
Dose of DMTU (mgg-1)
J " 150-
I
I
i 100-
h
0.5
D Saline
0DMTU
•5 200H
0
0.5
1.0
2.0
4.0
Dose of DMTU (mgg-1)
D Saline
E3DMTU
100Tl
50-
0.5
r
1.0
2.0
4.0
Dose of 0MTU (mgg"1)
times (15 min, 1 h, 4h, 16 h) before exposure to
66 % nitrous oxide in oxygen for 1 h. DMTU
impeded nitrous oxide-induced enzyme inactivation in liver (fig. 3 A) and kidney (fig. 3 B) for as
long as 4 h following injection, but the protective
effect of DMTU disappeared by 16 h in these
organs In brain (fig. 3 c), methionine synthase
activities were greater at all times following
DMTU injection compared with saline-injected
animals, but a significant difference was obtained
only when DMTU was injected 16 h before
exposure to nitrous oxide. Serum concentrations
of DMTU were similar (3-4 mg ml"1) in mice
injected 15 min to 4 h before exposure to nitrous
oxide, and decreased by more than 50 % in those
mice injected 16 h before exposure (table I). As in
the previous series of experiments, in animals not
exposed to nitrous oxide, DMTU had no significant effect on methionine synthase activity in
any of the organs examined (fig. 3 A, B, C).
Recovery of methionine synthase activity fol-
FIG. 2. Methionine synthase activities in livers (A), kidneys
(B) and brains (c) of mice injected with varying doses of
DMTU or an equivalent volume of saline 1 h before exposure
to 66% nitrous oxide in oxygen for 1 h. The circles indicate
mice that were not exposed to nitrous oxide. Each point was
determined from four separate animals. Error bars indicate
SD. Activities were compared between the DMTU- and
saline-injected mice for each DMTU dose, and between the
DMTU- and saline-injected mice that were not exposed to
nitrous oxide. Significance was calculated using an unpaired
t test: *P < 0.05; **P < 0.01; ***P < 0.001.
lowing inactivation by nitrous oxide was determined in a fourth series of experiments. In the
saline-injected mice, recovery of enzyme activity
occurred gradually over a 4-day period in liver,
kidney and brain (fig. 4A, B, C). In mice injected
with DMTU, recovery of methionine synthase
activity tended to be slower in all three organs.
DMTU produced a sedative effect that increased with increasing doses of the agent. At the
greatest dose (4 mg g"1) all of the animals lost
their righting reflex within 1 h of injection. At the
lowest dose (0.5 mg g"1) marked behavioural
changes were not evident. At the intermediate
DMTU dose used in many of the experiments (2
mg g"1) the animals were noticeably sedated and a
loss of the righting reflex was often observed.
When DMTU was administered alone, no toxic
effects were evident, and the sedative effects were
reversible. Mice were active 16 h after injection
with DMTU 2 mg g"1 (as performed in the third
series of experiments), and appeared only slightly
DIMETHYLTHIOUREA, METHIONINE SYNTHASE AND N2O
"o. 2 0 0 -
219
„<=> 3 0 0 I I Saline
NO N2O
D Saline
^ DMTU
^
2 m g
g-1
2 0 0 - NON2O
**
100-
DMTU 2 mg g" 1
100-
15min
15min
1h
4h
16h
Time of DMTU injection before N 2 0
~ 150-1
1h
4h
16 h
Time of DMTU injection before N 2 0
• Saline
2mgg" 1
~ 100-
ii
***
NON2O
50-
15min
1h
4h
16h
Time of DMTU injection before N20
TABLE I. Time dependence of serum DMTU concentrations in
mice. Mice were given 2.0 mg g~l i.p. injections of DMTU and
allowed to remain in room air for 15 min or 1, 4 or 16 h before
being exposed to 66% nitrous oxide for 1 h. After the 1-h
exposure, animals were killed immediately with 100% carbon
dioxide and the blood removed and organs isolated. Each of the
values below was obtained from four separate mice
FIG. 3. Methionine synthase activities in livers (A), kidneys
(B) and brains (c) of mice injected with DMTU 2 mg g"1 (or
an equivalent volume of saline) 15min, 1, 4 or 16 h before
exposure to 66 % nitrous oxide in oxygen for 1 h. The circles
indicate mice that were not exposed to nitrous oxide. Each
point was determined from four separate animals. Error bars
indicate SD. Activities were compared between the DMTUand saline-injected mice at each time point, and between the
DMTU- and saline-injected mice that were not exposed to
nitrous oxide. Significance was calculated using an unpaired
t test: *P < 0.05; **P < 0.02; ***P < 0.005.
injection. In addition, one of the mice in the 2-day
recovery group (fig. 4) died of unknown causes
between 1 and 2 days after exposure to nitrous
oxide and injection of DMTU.
DISCUSSION
Time of
DMTU injection
before N2O
Serum DMTU
concn (SD)
(mg ml"1)
Earlier studies have shown that the breakdown of
nitrous oxide via physical or biological mechanisms may be associated with the formation of
15 min
3.97 (0.34)
free radicals. The presence of nitrous oxide
3.16(0.57)
1h
markedly enhances the production of hydroxyl
4h
3.46(0.41)
radicals in irradiated aqueous solutions [15, 26].
16 h
1.46(0.31)
In human intestinal contents, metabolism of
nitrous oxide occurs via a reductive pathway [27],
"slower" than the saline-injected animals. How- and the use of a "spin trap" demonstrates that
ever, the sedative effect appeared to persist when this metabolism is associated with the production
injected immediately after a 4-h exposure to 66 % of free radicals [28].
nitrous oxide (as in the fourth series of experiThe possibility that nitrous oxide might inments); these animals appeared mildly sedated activate methionine synthase through the geneven 2 days (but not 4 days) after DMTU eration of a free hydroxyl radical at the active site
BRITISH JOURNAL OF ANAESTHESIA
220
J» 200-
-\» 300-
I
NON2O
NON^
• Saline
DSaline
1
200 H
2mgg"
100-
2mgg"1
***
100o
a>
Oh
6h
16h
2days
4days
Recovery time after exposure to N2O
U_
_6h....
16h
2days
Recovery time after exposure to N 2 0
4days
~ 150-,
• Saline
J 100- N0N 2 0
^
DMTU 2 mg g"1
•a
•Ei
**
50CO
c
'c
o
1
0-
0h
6h
16h
2 days
Recovery time after exposure to N2O
4 days
of the enzyme is supported by in vitro biochemical
studies [12, 13]. The isolated and nitrous oxideinactivated enzyme exhibits absorption and electron paramagnetic resonance spectra indicative of
cob(II)alamin, consistent with the scheme shown
in equation (1). As the hydroxyl radical is a
powerful oxidant that combines with most biological molecules at a rate that is nearly diffusioncontrolled [14, 29], "OH must react at or very
close to its site of formation (i.e., at the cofactor or
the substrate binding sites). Covalent modifications of the active site of methionine synthase
by reaction with 'OH provide a possible explanation for the essentially irreversible inhibition
of this enzyme by nitrous oxide.
Our objective was to test for the possible role of
hydroxyl radicals in inactivating methionine synthase by examining whether or not a scavenger of
hydroxyl radicals could protect the enzyme from
inactivation by nitrous oxide. DMTU was used
because it is a highly efficient scavenger of
FIG. 4. Methionine synthase activities in livers (A), kidneys
(B) and brains (c) of mice injected with DMTU 2 mg g~' (or
an equivalent volume of saline) immediately after exposure to
66 % nitrous oxide in oxygen for 4 h. Mice were returned to
a room air environment and killed with 100% carbon dioxide
at 0, 6 or 16 h, or 2 or 4 days following exposure to nitrous
oxide. The circle indicates mice that were not exposed to
nitrous oxide. Each point was determined from four separate
animals, with the exception of the DMTU-treated mice in the
2 day recovery group, in which one of the mice died. Error
bars indicate SD. Activities were compared between the
DMTU- and saline-injected mice at each time point, and
significance was calculated using an unpaired t test: *P <
0.05; **P < 0.02; ***P < 0.001.
hydroxyl radicals [15]; it penetrates into the
cytoplasm of cells [15] (where methionine synthase is located), and it protects various organs
against the injury associated with the production
of free radicals [15-22, 30]. The i.p. administration of DMTU slowed, but did not prevent,
the inactivation of methionine synthase by nitrous
oxide in three different organs (figs 1, 2). We
speculate that a delay in the inactivation occurs
when DMTU molecules near the active site of the
enzyme scavenge some (but not all) of the
hydroxyl radicals produced by reaction of vitamin
B l2 with nitrous oxide. The higher enzyme
activities in the DMTU-treated animals compared with the saline-treated animals exposed to
nitrous oxide cannot be explained by an effect of
DMTU on methionine synthase activity per se, as
the administration of DMTU to animals not
exposed to nitrous oxide did not alter enzyme
activity (figs 2, 3).
The ability of DMTU 2 mg g"1 to protect
DIMETHYLTHIOUREA, METHIONINE SYNTHASE AND N2O
methionine synthase against inactivation by nitrous oxide occurred within 15 min of injection
and persisted for 4 h or longer (fig. 3). The
duration of the protective effect of DMTU
appeared to be longer in brain (fig. 3 c) than liver
(fig. 3A) or kidney (fig. 3 B). The prolonged effect
of DMTU is consistent with the serum half-life of
DMTU in the mouse, which is 5-17 h (table I).
This half-life of DMTU in the mouse serum is
shorter than the plasma half-lives (approximately
25 h) measured in rats [15], dogs [22], and sheep
[31] given DMTU at doses ranging from 0.5 to
0.75 g kg"1.
If the inactivation of methionine synthase by
nitrous oxide proceeds via the mechanism in
equation (1), the generation of a hydroxyl radical
at the active site of the enzyme might react
covalently with amino acid residues, vitamin B12,
or both. In this process, the hydroxyl radical
would be consumed and the enzyme damaged
permanently. Thus if DMTU were given after
the irreversible reaction of the hydroxyl radical
with the active site of the enzyme, it should not
enhance recovery of enzyme activity. Indeed,
DMTU did not enhance recovery of methionine
synthase activity following nitrous oxide inactivation, but instead slightly delayed enzyme
recovery compared with saline-injected mice (fig.
4 A, B, C). Methionine synthase activities in livers,
kidneys and brains of saline-injected animals
gradually recovered over the 4-day period after
nitrous oxide inactivation, consistent with previous results in mice [2, 10]. The reason for the
slower recovery in the DMTU-treated animals
during the first day after exposure to nitrous oxide
is not known, but one possibility is that DMTU
may impair protein synthesis, and that synthesis
of new enzyme is required for recovery of
activity.
The present studies also suggest an explanation
for the marked differences in the time course of
methionine synthase inhibition between species
and between different organs in the same species.
For example, rats exposed to 50% nitrous oxide
exhibit rapid inactivation of hepatic methionine
synthase with a half-time of approximately 5 min
[5], whereas the rate of inactivation of hepatic
methionine synthase activity in patients exposed
to 50-70 % nitrous oxide is of the order of 1 or 2 h
[4, 5]. Similarly, the rate of enzyme inactivation
in mice exposed to 66 % nitrous oxide is slower in
brain (fig. 1C) than in liver (fig. 1 A). If methionine
synthase inactivation by nitrous oxide results
221
from the production of a hydroxyl radical (equation (1)), the presence of intrinsic cytoplasmic
antioxidants capable of scavenging hydroxyl radicals might slow this inactivation. A slower
inactivation rate in human liver compared with rat
liver and in mouse brain compared with mouse
liver might be associated with relatively high
concentrations of intrinsic hydroxyl radical scavengers in these organs.
The present findings demonstrate that DMTU
impeded the inactivation of methionine synthase
by nitrous oxide and are consistent with the
hypothesis that a hydroxyl radical is produced
when nitrous oxide reacts with the Co+1 atom of
vitamin B12 and that this hydroxyl radical damages
the enzyme. However, other interpretations
should be considered. A delay induced by DMTU
in the equilibration of nitrous oxide seems an
unlikely explanation, as the partial pressure of
nitrous oxide in vessel-rich organs approaches
that of the inspired partial pressure within several
minutes after nitrous oxide administration [32].
The possibility also exists that DMTU might
impair the penetration of nitrous oxide molecules
to vitamin B12. This could occur directly by
surrounding the cofactor with relatively high
concentrations of DMTU molecules, or indirectly
by a DMTU-induced conformational change in
the enzyme, thereby resulting in a vitamin B12
molecule that is less accessible to nitrous oxide.
Another possible explanation is that DMTU may
decrease enzyme turnover in vivo, as methionine
synthase inactivation by nitrous oxide apparently
occurs only while the enzyme is turning over [12].
Finally, it should be noted that, while DMTU is
an effective scavenger of hydroxyl radicals, it is
also an effective scavenger of other oxidants [33],
and that its protective effect is not proof of
damage mediated by a hydroxyl radical.
Through inactivation of methionine synthase,
nitrous oxide might contribute to the haematological and neurological abnormalities seen
occasionally in patients following anaesthesia [34].
As DMTU only slows the enzyme inactivation
produced by nitrous oxide and does not prevent
inactivation (fig. 1), and as DMTU needs to be
given in relatively high doses before administration of nitrous oxide to impede inactivation, it
is unlikely that DMTU will be useful clinically to
counteract the effects of nitrous oxide. Furthermore, the long-acting sedative properties of
DMTU (also noted by others [31]; Michael T.
Snider, personal communication), provide an
222
additional difficulty in the clinical use of this
compound.
BRITISH JOURNAL OF ANAESTHESIA
15. Fox RB. Prevention of granulocyte-mediated oxidant lung
injury in rats by a hydroxyl radical scavenger, dimethylthiourea. Journal of Laboratory Investigation 1984; 74:
1456-1464.
ACKNOWLEDGEMENTS
16. Treda LS, Beehler CJ, Banerjee A, Brown JM, Grosso
This research was supported in part by a Merit Review Grant
MA, Harken AH, McCord JM, Repine JE. Hyperoxia
from the Veterans Administration, by NIA Grant PO1
and self- or neutrophil-generated O2 metabolites inAG3104, by a grant from the Academic Senate of the
activate xanthine oxidase. Journal of Applied Physiology
University of California at San Francisco, and by the UCSF
1988; 65: 2349-2353.
Anesthesia Research Foundation.
17. Jackson RM, Veal CF, Alexander CB, Brannen AL,
Fulmer JD. Re-expansion pulmonary edema. A potential
role for free radicals in its pathogenesis. American Review
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