61, 273–282 (2001)
Copyright © 2001 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Effects of Respiratory Acidosis and Alkalosis on the Distribution
of Cyanide into the Rat Brain
Amina Djerad,* Claire Monier,* Pascal Houzé,† Stephen W. Borron,* ,‡
Jeanne-Marie Lefauconnier,* and Frédéric J. Baud* ,1
*INSERM U26, Université Paris 7, Hôpital Fernand Widal, 200 rue du Faubourg Saint-Denis, 75010 Paris, France; †Laboratoire de Biochimie A,
Hôpital Saint-Louis, 1 avenue Claude Vellefaux, 75010 Paris, France; and ‡Department of Emergency Medicine,
George Washington University Hospital, 901 23rd Street NW, Washington, DC 20037
Received November 17, 2000; accepted February 23, 2001
The aim of this study was to determine whether respiratory
acidosis favors the cerebral distribution of cyanide, and conversely, if respiratory alkalosis limits its distribution. The pharmacokinetics of a nontoxic dose of cyanide were first studied in a
group of 7 rats in order to determine the distribution phase. The
pharmacokinetics were found to best fit a 3-compartment model
with very rapid distribution (whole blood T 1/2␣ ⴝ 21.6 ⴞ 3.3 s).
Then the effects of the modulation of arterial pH on the distribution of a nontoxic dose of intravenously administered cyanide into
the brains of rats were studied by means of the determination of
the permeability-area product (PA). The modulation of arterial
blood pH was performed by variation of arterial carbon dioxide
tension (PaCO 2) in 3 groups of 8 anesthetized mechanically ventilated rats. The mean arterial pH measured 20 min after the start
of mechanical ventilation in the acidotic, physiologic, and alkalotic groups were 7.07 ⴞ 0.03, 7.41 ⴞ 0.01, and 7.58 ⴞ 0.01,
respectively. The mean PAs in the acidotic, physiologic, and alkalotic groups, determined 30 s after the intravenous administration
of cyanide, were 0.015 ⴞ 0.002, 0.011 ⴞ 0.001, and 0.008 ⴞ 0.001
s –1, respectively (one-way ANOVA; p < 0.0087). At alkalotic pH
the mean permeability-area product was 43% of that measured at
acidotic pH. This effect of pH on the rapidity of cyanide distribution does not appear to be limited to specific areas of the brain. We
conclude that modulation of arterial pH by altering PaCO 2 may
induce significant effects on the brain uptake of cyanide.
Key Words: cyanide; pharmacokinetics; respiratory acidosis; respiratory alkalosis; brain.
Cyanide poisoning may result from exposure to hydrocyanic
acid and cyanide salts (Benaissa et al., 1995; Graham et al.,
1977) as well as to various cyanogenic compounds including
sodium nitroprusside (Vesey, 1987) and nitriles (Bismuth et
al., 1987; Caravati et al., 1988). However, the most frequent
circumstance associated with cyanide poisoning is smoke inhalation (Anderson and Harland, 1982; Ballantyne, 1974; Baud
1
To whom correspondence should be addressed at Réanimation Médicale et
Toxicologique, Hôpital Lariboisière, Université Paris 7, 2 rue Ambroise Paré,
75010 Paris, France. Fax: (033)-1-49-95-65-78. E-mail: [email protected].
et al., 1991; Clark et al., 1981; Silverman et al., 1988). Cyanide is produced by the thermal degradation of nitrogen-containing products found in numerous common household materials (Alarie, 1985; Ballantyne, 1974; Yamamoto, 1975;
Yamamoto and Yamamoto, 1971). Smoke-induced cyanide
intoxication is always associated with the inhalation of other
gases. The role of the association of carbon monoxide with
cyanide has been extensively studied (Baud et al., 1991; Hartzell, 1989; Moore et al., 1991; Tsuchiya, 1989). In contrast, the
role of carbon dioxide has only been studied to a very limited
extent. However, fires always produce enormous quantities of
carbon dioxide, reaching thousands of parts per million in fire
atmospheres (Hartzell, 1989).
Inhalation of a high concentration of carbon dioxide induces
a profound and immediate effect on the pattern of breathing.
Carbon dioxide immediately increases minute ventilation
(Read, 1967), contributing to the overall hazard of fire gas
environment by causing accelerated lung absorption of gaseous
toxicants (Hartzell, 1989). However, the further contribution of
carbon dioxide to the systemic toxicity of cyanide has not been
addressed. Acute retention of carbon dioxide provokes respiratory acidosis, which dramatically augments cerebral blood
flow (Feihl and Perret 1994; Hossmann, 1990; Hossmann and
Blöink, 1981; Kety and Schmidt, 1948). Furthermore, respiratory acidosis can significantly modify the ratio of nonionized to
ionized forms of xenobiotics, depending on the pH of the
milieu and the pK a of the substance (Rowland and Tozer,
1995). Accordingly, respiratory acidosis has been shown to
increase the tissue distribution of weak acids such phenobarbital (Waddell and Butler, 1957) and salicylate (Hill, 1973).
Hydrocyanic acid is a weak acid with a pK a of 8.99 at 35°C
(Izatt et al., 1962). The olive oil/water partition coefficient of
cyanide is 0.15 and the brain uptake index is 41 ⫾ 4% (Oldendorf, 1974). Thus, hydrocyanic acid is considered a lipophilic substance (Oldendorf, 1974). The movement of cyanide through membranes is complex. But accumulation of
labeled cyanide by neuronal tissue is not an energy-dependent
process (Borowitz et al., 1994). These data led us to the
273
274
DJERAD ET AL.
hypothesis that respiratory acidosis might increase the distribution of cyanide into the brain by increasing the amount of
toxicant delivered to the brain by the bloodstream and by
increasing the nonionized fraction. Conversely, respiratory alkalosis might be expected to have opposite effects on the
distribution of cyanide into the brain.
The effect of modulation of arterial pH by ventilation is also
of importance when considering the supportive treatment of
acute cyanide poisoning. Mechanical controlled ventilation is
the basic standard of care of severe cyanide poisoning (Ellenhorn et al., 1997). Oxygen has been shown to significantly
counteract the toxicity of cyanide in spontaneously breathing
rats poisoned with cyanide (Burrows et al., 1973; Isom and
Way, 1974). But to our knowledge, no study has addressed the
effects of modulation of arterial pH through controlled ventilation on either the toxicokinetics or the toxicity of cyanide.
The question to be addressed is whether respiratory acidosis
favors the cerebral distribution of cyanide, and conversely, if
respiratory alkalosis limits its distribution. To answer this
question, we first determined the pharmacokinetics of a nontoxic dose of cyanide in rats in order to determine the distribution phase. Then, we studied the effects of the modulation of
arterial pH induced by controlled ventilation on the distribution
of a nontoxic dose of cyanide administered intravenously into
the brains of anaesthetized rats.
An isotopic dosage of cyanide in whole blood was performed, based on a
modification of the classic microdiffusion method in a Conway cell (Prieux,
Joinville-le-Pont, France). This method is based on the liberation of blood
cyanide in the form of hydrogen cyanide gas by the action of sulfuric acid in
the outer chamber of the Conway cell. The HCN produced is captured by
sodium hydroxide in the inner chamber (Rieders, 1975). Five hundred ␮l of 0.5
N sodium hydroxide were placed in the inner chamber of the Conway cell.
Three hundred ␮l of normal saline and 500 ␮l of sulfuric acid 0.1 N were
placed in the external chamber. A 200-␮l blood sample was also placed in the
outer chamber, remaining out of contact with the acid until the chamber was
sealed hermetically. Microdiffusion was complete after 2 h at ambient temperature. The counting of radioactivity was performed on 20 ␮l of the sodium
hydroxide phase contained in the internal chamber added to 10 ml of scintillation liquid (Pico-Fluor, Packard, France). A Tri-carb counter (Model 1900
TB, Packard) was used to measure the radioactivity. All results were corrected
for background radiation.
As the method of measurement of blood cyanide concentrations is specific
for cyanide and not affected by cyanide metabolites, the results of the radioactivity were transformed into nmol/l, taking into account the specific activity
(56 mCi/mmol) of the radiolabeled cyanide. The modeling of the kinetics of
nontoxic doses of cyanide in blood was performed using the Siphar software
program (Simed, Créteil, France).
MATERIALS AND METHODS
● Acid pH: hypoventilation using carbogen (93% O 2 , 7% CO 2 , Air Liquide),
Vt ⫽ 2 ml/kg, frequency ⫽ 75/min;
● Physiologic pH: normal ventilation on pure oxygen, Vt ⫽ 8 ml/kg,
frequency ⫽ 75/min;
● Alkaline pH: hyperventilation on pure oxygen, Vt ⫽ 12.5 ml/kg, frequency ⫽ 75/min.
Animals. Sprague-Dawley OFA rats weighing between 300 and 350 g
(9 –10 weeks of age) were obtained from Iffa Credo (Domaine des Arbresles,
69592 L’Arbresles, France). Animals were housed in a light- and temperaturecontrolled setting with access to food and water ad libitum. All experiments
were carried out within the ethical guidelines established by the National
Institutes of Health and the French Minister of Agriculture.
Preparation of the solution of radioactive cyanide. One mCi of 14Clabeled potassium cyanide (New England Nuclear, Amersham, MA; specific
activity: 56 mCi/mmol) was diluted in 40 ml of NaOH 0.1N and stored at
–20°C. Cyanide salts autodegrade to carbonates (Christel et al., 1977), a
process accelerated in the presence of water. Thus, in order to precipitate
14
CO 3 2–, an aliquot of the parent solution was treated immediately before use,
as follows (Christel et al., 1977; McMillan and Svoboda, 1982). Five hundred
␮l was thawed and then precipitated with 30 ␮l of BaCl 2 3 M and 20 ␮l of
NaHCO 3 0.1 N. The mixture was vortexed, then centrifuged at 5000 rpm for
10 min. The supernatant was then decanted and used for the studies.
Kinetics of radiolabeled cyanide in whole blood. Seven animals were
anesthetized with Ketamine (Ketalar威, 70 mg/kg; Parke-Davis, France) and
diazepam (Valium威, 7.5 mg/kg; Roche, France) ip, then placed on a warming
blanket with a regulating thermostat. A rectal probe permitted feedback control
of the temperature. The femoral artery and vein were catheterized, the venous
catheter permitting systemic heparinization of the animal, as well as administration of the labeled cyanide. The arterial catheter permitted blood collection
for the quantification of blood cyanide. Following catheterization, animals
were heparinized with 500 ␮l/kg body weight of 10% heparinized saline. Five
min after heparinization, 200 ␮l of purified 14C-labeled potassium cyanide,
corresponding to 160 to 187 nmol CN –/kg body weight, were administered as
an intravenous bolus. These doses are dramatically below the reported toxic
doses in the rat (Ballantyne, 1987). Three hundred ␮l of arterial blood were
then collected at 30, 60, and 90 s and at 2, 6, 10, 30, 60, and 90 min.
Effects of Respiratory Acidosis and Alkalosis on the Distribution of Cyanide
into the Rat Brain
Development of a model of acidification and alkalinization. This model
of respiratory acidification and alkalinization is based on the ventilation of
animals under neuromuscular blockade at various tidal volumes (Vt) and
constant frequency. For realization of the model, 3 modes of ventilation were
used:
The animals were anesthetized in a manner identical to that previously
described, using ketamine and diazepam. The temperature of the animals was
continuously monitored. The femoral artery and vein were catheterized and the
animal subsequently tracheotomized and ventilated using a small animal
ventilator (Harvard Biosciences, France). The animals in the acid pH group
were ventilated with carbogen. Otherwise, animals received pure oxygen (Air
Liquide). A preliminary study using repeated measures of blood gases of
animals submitted to various ventilatory schemes demonstrated that alkalinization or acidification was achieved based on the Vt chosen. This modification
of blood pH reached a plateau at 15 min, both for those animals with induced
respiratory acidosis, as well as those placed in respiratory alkalosis. The
modification of pH was stable for at least 15 min. Thus, a 500 ␮l blood
specimen was collected for blood gas determination in a heparinized syringe
(Bayer Diagnostics, France) 20 min after the start of mechanical ventilation.
Arterial blood gases were measured by means of a blood gas analyzer
(Radiometer, Copenhagen, Denmark).
Determination of cyanide distribution by the method of Ohno. This
method, which studies the cerebral distribution of radioactive compounds, was
undertaken using animals previously ventilated to obtain variable conditions of
blood pH (alkaline, physiologic, or acid). The technique is composed of
injecting the radioactive cyanide, along with tritiated sucrose, which permits
the determination of the volume of the vascular space (Ohno et al., 1978).
We used the results of the previously performed pharmacokinetics study in
order to determine the time for decapitation of the animal. The animals were
prepared as previously stated. An aspirating pump was connected to the arterial
catheter. Twenty min after the beginning of controlled ventilation an arterial
275
ARTERIAL pH AND CYANIDE DISTRIBUTION
blood gas was collected to verify the pH conditions, just before the administration of the radioactive cyanide and sucrose. The radioactive products were
administered as a 200-␮l iv bolus of normal saline containing 20 ␮Ci of
tritiated sucrose and 400 ␮l of purified 14C-cyanide equivalent to 6.25 ␮Ci
corresponding to 320 to 373 nmol CN –/kg body weight. Arterial blood was
drawn by the pump at a constant aspiration rate of 0.4 ml/min for a fixed time
of 30 s from the end of injection of radioactive compounds until decapitation.
Decapitation of the animal occurred 30 s after injection of the cyanide, as this
represents 1.5 times the distribution half-life, as determined in the kinetic
study. Two aliquots of the arterial blood were immediately centrifuged to
separate plasma from erythrocytes. Five hundred ␮l of blood were obtained
from the neck using a micropipette for determination of tritiated sucrose
activity. The brain was immediately removed from the cranium and placed on
crushed ice. The arachnoid and subarachnoid membranes were delicately
removed. The brain was dissected into various parts, namely: pons-medulla,
cerebellum, superior and inferior colliculi, hypothalamus, thalamus, hippocampus, striatum, frontal cortex, parietal cortex, and occipital cortex. These structures were placed in flasks and weighed, followed by addition of 1 ml of
Soluene (Packard), then sealed. The tissues were digested in a water bath at
60°C for 2 h. The tubes were counted after addition of 9 ml of scintillation
liquid. 14C and 3H activities were measured in all brain tissue specimens. Blood
and plasma samples of 20 ␮l were placed in flasks and weighed, followed by
addition of 1 ml of Soluene and then of 9 ml of scintillation liquid (Packard),
sealed and counted. This treatment was performed in duplicate, on arterial
blood for 14C and on blood collected at the neck immediately after decapitation
for 3H. All results were corrected for background radiation.
Ohno’s method permits the determination of cerebrovascular permeability
of nonelectrolytes in rats. The method requires the determination of the amount
of cyanide distributed into brain tissue. However, the amount of cyanide in
whole brain (Q c) is the sum of the amount of cyanide in the brain tissue (Q Br)
and the amount of cyanide contained in the blood vessels within the brain
structure (Q v). The amount of cyanide contained in the blood vessels within the
brain structure can be calculated given the regional blood volume.
Regional blood volume. In each brain structure, we measured the tritiated
sucrose activity (dpm/mg). We also measured the tritiated sucrose activity in
whole blood measured by means of the blood specimen collected at the neck after
decapitation (dpm/mg of blood). The regional vascular volume (Vr), expressed as
a percentage of the weight of the structure, is equal to the ratio of tritiated sucrose
activity in the brain structure to the tritiated sucrose activity in blood.
Amount of radiolabeled cyanide in the regional blood volume (Q v). The
amount of radiolabeled cyanide in the regional blood volume is equal to the
product of the regional blood volume and the activity of radiolabeled cyanide
in blood (Q blood):
Qv ⫽ Vr (%) ⫻ Qblood (dpm/mg)
Quantity of radiolabeled cyanide having penetrated into the brain (Q Br).
In each brain structure we measured the whole 14C-cyanide activity (Q c,
dpm/mg). The quantity of cyanide having penetrated into the brain is equal to:
QBr ⫽ Qc – Qv (dpm/mg)
Calculation of the permeability-area product. The calculation used in the
method of Ohno is that of the permeability-area product (PA). The PA is the
ratio between the quantity of the substance having penetrated into the brain
(Q Br) to the amount of drug delivered to the brain during the same time as
assessed by the integral of the arterial radioactivity.
冕
PA ⫽ QBr / Cp 䡠 dt
where 兰C p ⫽ activity measured in the arterial plasma recovered by aspiration
during the period of time (dt) after injection of the solution of the radiolabeled
FIG. 1. The pharmacokinetics in whole blood of a nonlethal dose of
radiolabeled cyanide administered as an intravenous bolus in 7 rats. Results are
expressed as mean ⫾ S.E.M.
product, representing the integral of the blood concentrations during the 30 s.
Results are expressed as s –1.
Statistical analysis. All comparisons were made using analysis of variance
(ANOVA), followed by multiple comparisons using Bonferonni’s correction
(Prism 2.0 Software, GraphPad, Inc., San Diego, CA). Results are expressed as
mean ⫾ SE. A p value less than 0.05 was considered significant.
RESULTS
Kinetics of a Nonlethal Dose of Cyanide in Whole Blood
The kinetic study of a nontoxic dose of radiolabeled cyanide
in whole blood was performed on 7 rats (Fig. 1). The best fit
was obtained using a 3-compartment model (Table 1). This
analysis revealed that the distribution of cyanide was extremely
rapid with a T 1/2␣ in whole blood of 21.6 ⫾ 3.3 s. The
elimination was relatively long, with a T 1/2␥ in whole blood of
87.4 ⫾ 22.9 min.
Effects of Respiratory Modulation on the Cerebral
Distribution of Cyanide
Modulation of arterial pH by the different modes of controlled ventilation. The mean arterial pH determined in arterial blood gases collected 20 min after the start of mechanical
ventilation is shown in Table 2. The comparison of arterial pH
revealed a significant difference between animals in the acidotic and physiologic groups (p ⬍ 0.001), the alkalotic and
physiologic groups (p ⬍ 0.001), and the acidotic and alkalotic
pH groups (p ⬍ 0.001).
The comparison of the mean arterial PaCO2 (Table 2) revealed
significant differences between animals in the acidotic and physiologic groups (p ⬍ 0.001), and the acidotic and alkalotic pH
groups (p ⬍ 0.001). The PaCO 2 between animals in the alkalotic
and physiologic groups did not differ significantly.
The comparison of the mean arterial bicarbonate (Table 2)
revealed significant differences between animals in the acidotic
276
DJERAD ET AL.
TABLE 1
Pharmacokinetics in Whole Blood of a Nontoxic Dose of 14C-labeled Cyanide Administered as a Bolus Dose in 7 Rats
1
2
3
4
5
6
7
Mean ⫾ SEM
Cmax (nmol/l)
T 1/2␣ (s)
T 1/2␤ (s)
T 1/2␥ (min)
Vd (l/kg)
Cltot (ml/min/kg)
983
749
879
604
837
1204
830
869 ⫾ 71
13
12
38
22
21
25
20
21.6 ⫾ 3.3
40
132
293
125
74
207
137
144.0 ⫾ 31.8
40
61
69
120
212
61
49
87.4 ⫾ 22.9
0.65
0.84
0.84
0.87
1.01
0.79
0.81
0.83 ⫾ 0.04
11.3
11.5
8.5
5.0
3.3
8.9
11.3
8.5 ⫾ 1.2
and physiologic groups (p ⬍ 0.05), the alkalotic and physiologic groups (p ⬍ 0.05), and the acidotic and alkalotic groups
(p ⬍ 0.001).
There were no significant differences in the mean PaO 2
between groups (Table 2).
Distribution of cyanide into rat blood, plasma, and brain.
Table 3 shows the mean regional blood volumes of the 10
structures in each group. There were no significant differences
when comparing the mean regional blood volumes measured in
each of the 10 brain structures in the acidotic, physiologic, and
alkalotic groups. The mean global blood volumes (mean of the
sum of regional blood volumes), expressed as a percentage of
the weight of the structures in the acidotic, physiologic and
alkalotic groups were 2.6 ⫾ 0.3, 2.4 ⫾ 0.1, and 2.5 ⫾ 0.1%,
respectively. There were no significant differences of the mean
global blood volumes in the 3 groups (Fig. 2).
The mean 14CN activities in blood samples collected at the
neck after decapitation in the acidotic, physiologic, and alkalotic groups were 626.2 ⫾ 159.0, 647.9 ⫾ 221.3, and 617.7 ⫾
159.5 dpm/mg of blood, respectively. There were no significant differences among the 3 groups.
The mean plasma 14CN activities in arterial samples collected by aspiration during 30 s in the acidotic, physiologic,
and alkalotic groups were 390.0 ⫾ 77.9, 337.3 ⫾ 96.2, and
477.1 ⫾ 104.1 dpm/mg of blood, respectively. There were no
significant differences among the 3 groups.
Table 4 and Figure 3 show the mean regional PAs of the 10
structures for each treatment group. The only significant difTABLE 2
Arterial Blood Gases in the Acidotic, Physiologic, and Alkalotic
Groups Just Before Administration of Cyanide
pH
PaCO 2 (mm Hg)
PaO 2 (mm Hg)
HCO 3⫺ (mmol/l)
Acidotic
Physiologic
Alkalotic
7.07 ⫾ 0.03
93.9 ⫾ 7.9
292.6 ⫾ 28.7
25.9 ⫾ 1.1
7.41 ⫾ 0.01
32.5 ⫾ 1.4
324.9 ⫾ 16.0
21.7 ⫾ 0.7
7.58 ⫾ 0.01
17.4 ⫾ 1.2
275.2 ⫾ 20.9
17.2 ⫾ 1.3
Note. Results are expressed as mean ⫾ SEM.
ference found was in the physiologic group where the PA of
the colliculi was significantly greater than in the hypothalamus
(0.017 ⫾ 0.003, 0.009 ⫾ 0.001 s –1, respectively; p ⬍ 0.05).
The mean global PA in the acidotic, physiologic, and alkalotic groups were 0.015 ⫾ 0.002, 0.011 ⫾ 0.001, and 0.008 ⫾
0.001 s –1, respectively (Fig. 4). ANOVA showed a significant
difference in the mean global PAs for the different groups (p ⫽
0.0087). Post hoc testing revealed that the mean PA of animals
at acid pH was significantly greater than the mean global PA of
animals at alkaline pH (p ⬍ 0.01). There were no significant
differences between the mean global PAs of physiologic and
acid pH groups or between physiologic and alkalotic pH
groups.
For each of the 10 isolated brain structures, the mean regional PAs were compared among treatment groups (Table 4).
ANOVA and post hoc testing revealed that the mean regional
PAs of animals at acid pH were significantly greater than those
of animals at alkaline pH (Fig. 5) for the following structures:
pons-medulla (p ⬍ 0.01), hypothalamus (p ⬍ 0.05), thalamus
(p ⬍ 0.05), hippocampus (p ⬍ 0.05), frontal cortex (p ⬍ 0.05),
parietal cortex (p ⬍ 0.05), and occipital cortex (p ⬍ 0.05). In
the cerebellum, colliculi, and striatum, although the regional
TABLE 3
Regional Blood Volumes in the 10 Cerebral Structures in the
Acidotic, Physiologic, and Alkalotic Groups
Pons-medulla
Cerebellum
Colliculi
Hypothalamus
Thalamus
Hippocampus
Striatum
Frontal cortex
Parietal cortex
Occipital cortex
Acidotic (%)
Physiologic (%)
Alkalotic (%)
3.9 ⫾ 0.5
3.4 ⫾ 0.7
2.4 ⫾ 0.4
3.1 ⫾ 0.5
2.8 ⫾ 0.6
2.5 ⫾ 0.5
1.3 ⫾ 0.2
2.1 ⫾ 0.1
2.1 ⫾ 0.2
3.4 ⫾ 0.9
3.6 ⫾ 0.1
3.6 ⫾ 0.4
2.6 ⫾ 0.3
3.0 ⫾ 0.6
1.6 ⫾ 0.2
1.8 ⫾ 0.1
1.2 ⫾ 0.1
1.9 ⫾ 0.2
2.4 ⫾ 0.2
2.2 ⫾ 0.3
3.7 ⫾ 0.1
2.6 ⫾ 0.5
2.2 ⫾ 0.3
2.7 ⫾ 0.3
2.6 ⫾ 0.7
1.9 ⫾ 0.3
1.5 ⫾ 0.2
2.4 ⫾ 0.2
2.4 ⫾ 0.2
2.6 ⫾ 0.2
Note. Regional blood volumes are expressed as the percentage of the weight
of the structure. Results are expressed as mean ⫾ SEM.
277
ARTERIAL pH AND CYANIDE DISTRIBUTION
FIG. 2. The mean global vascular volumes in the brain according to
treatment groups. There were no significant differences of the mean global
blood volumes in the 3 groups.
PAs in the acidotic group were consistently greater than those
in the alkalotic group, the differences were not statistically
significant. The results in the physiologic group did not differ
statistically from either the acidotic or alkalotic groups.
DISCUSSION
The pharmacokinetics of both nontoxic and toxic doses of
cyanide administered intravenously to animals are poorly understood, with published studies essentially limited to dogs
(Bright and Marrs, 1988; Marrs and Bright, 1987; Sylvester et
al., 1983). Pharmacokinetic studies of cyanide have generally
shown that the decay of blood cyanide concentrations approximates first-order monocompartmental kinetics when blood
specimens are followed for 60 to 120 min after injection
(Bright and Marrs, 1988; Marrs and Bright, 1987; Sylvester et
al., 1983). However, when the decay of blood cyanide concentrations was followed for 245 min, a second phase was observed, consistent with a slow elimination of cyanide from the
blood of untreated dogs (Bright and Marrs, 1988). The late
half-life in whole blood in dogs poisoned with cyanide but not
treated with an antidote varies tremendously depending on
whether this second elimination phase is taken into account.
Thus, Marrs and Bright reported half-lives in whole blood of
18.4 min (1987) and 24 min (1988) for the first phase and 5.5 h
(1988) for the second phase. Our data, collected in untreated
rats having received a nontoxic dose of cyanide, are consistent
with a 3-phase decay of blood cyanide concentration. In agreement with others, we performed the kinetic study using whole
blood. It should be pointed out that the method we used
measured native cyanide and not its metabolites.
Numerous methodological differences may explain the discrepancies between our results and those previously reported.
While rats are frequently used to assess the toxicity of cyanide,
the toxicokinetics have not been reported. The studied species
may account for some discrepancies. Furthermore, in contrast
with others, we performed repeated blood sampling in the early
phase after the iv injection. A previous study performed in
mice using a nontoxic dose of labeled cyanide showed that
tissue distribution, assessed by means of whole-body autoradiography, was largely complete by as early as 5 min after iv
administration (Clemedson et al., 1960). We observed a very
rapid distribution of cyanide with a mean T 1/2 in whole blood
of about 22 s, which is consistent with the equally rapid onset
of cyanide toxicity.
The reported apparent volume of distribution of cyanide
varies according to species and among investigators: 0.075 l/kg
in humans (Schulz et al., 1982), and 0.209 l/kg (Bright and
Marrs, 1988) or 0.498 l/kg in dogs (Sylvester et al., 1983).
However, these apparent volumes of distribution appear rather
low, suggesting a very limited tissue penetration of cyanide.
Indeed, the previous reported apparent volumes of distribution
are consistent with a sequestration of cyanide partly in the
intravascular space (Bright and Marrs, 1988; Sylvester et al.,
1983) or extracellular compartment (Sylvester et al., 1983). In
contrast, we found a larger apparent volume of distribution
(i.e., 0.83 l/kg) than previously reported. We believe that our
data are more consistent with the lipophilic properties of cyanide (Oldendorf, 1974), and with its intracellular action at a
mitochondrial level in many tissues.
Variations in the pH of blood or urine influence the pharmacokinetics of numerous xenobiotics including salicylate
(Cumming et al., 1964; Goldberg et al., 1961; Hill, 1973;
Prescott et al., 1982), barbital (Brodie et al., 1960), phenobarbital (Goldberg et al., 1961; Waddell and Butler, 1957), acetazolamide (Goldberg et al., 1961), pentachlorophenol (Uhl et
al., 1986), hexachlorophene (Flanagan et al., 1995), and chlorophenoxy herbicides such as 2,4-dichlorophenoxyacetic acid,
and 4-chloro-2-methylphenoxypropionic acid (Flanagan et al.,
1990; Prescott et al., 1979).
Modulation of arterial pH alters not only the renal excretion
but also the tissue distribution of weak acids, whether modified
TABLE 4
Regional Permeability-Area Products for Cyanide in the 10
Cerebral Structures in the Acidotic, Physiologic, and Alkalotic
Groups
Pons-medulla
Cerebellum
Colliculi
Hypothalamus
Thalamus
Hippocampus
Striatum
Frontal cortex
Parietal cortex
Occipital cortex
Acidotic (s –1)
Physiologic (s –1)
Alkalotic (s –1)
0.015 ⫾ 0.001
0.012 ⫾ 0.002
0.016 ⫾ 0.003
0.015 ⫾ 0.002
0.015 ⫾ 0.002
0.014 ⫾ 0.002
0.014 ⫾ 0.002
0.013 ⫾ 0.002
0.012 ⫾ 0.001
0.017 ⫾ 0.004
0.012 ⫾ 0.001
0.012 ⫾ 0.001
0.017 ⫾ 0.003
0.009 ⫾ 0.002
0.011 ⫾ 0.001
0.012 ⫾ 0.001
0.013 ⫾ 0.001
0.010 ⫾ 0.001
0.009 ⫾ 0.001
0.011 ⫾ 0.001
0.008 ⫾ 0.001
0.006 ⫾ 0.001
0.008 ⫾ 0.001
0.008 ⫾ 0.001
0.009 ⫾ 0.001
0.007 ⫾ 0.001
0.009 ⫾ 0.001
0.007 ⫾ 0.001
0.007 ⫾ 0.001
0.007 ⫾ 0.001
Note. Results are expressed as mean ⫾ SEM.
278
DJERAD ET AL.
FIG. 4. The mean permeability-area products (mean PA) in the brain
according to treatment groups. The mean PA in the acidotic, physiologic and
alkalotic groups were 0.015 ⫾ 0.002, 0.011 ⫾ 0.001, and 0.008 ⫾ 0.001 s –1,
respectively. The mean PA of animals at acid pH was significantly greater than
the mean global PA of animals at alkaline pH (p ⬍ 0.01). There were no
significant differences between the mean global PAs of physiologic and acid
pH groups or between physiologic and alkalotic pH groups.
via metabolic (Brodie et al., 1960; Flanagan et al., 1995;
Waddell and Butler, 1957) or respiratory (Goldberg et al.,
1961; Hill, 1973; Waddell and Butler, 1957) mechanisms. The
effects of the modulation of arterial pH on the brain tissue
distribution in these studies was assessed by measuring the
ratio of brain to plasma concentrations. While the degree of
modulation may vary with the mechanism of induction, the end
result is predictable: an increase in arterial pH decreases brain
tissue distribution while acidification has the opposite effect.
Toxic doses of cyanide are known to cause both intracellular
acidosis (Lotito et al., 1989) and acidemia (Dodds et al., 1992;
Katsumata et al., 1980; Salkowski and Penney, 1995). Furthermore, a toxic dose of cyanide has been shown to significantly
increase the blood-brain barrier permeability (Olesen, 1986).
Thus, in order to study the effects of respiratory acidosis/
alkalosis per se, we chose to study the distribution of a nontoxic dose of cyanide.
We measured the permeability-area product of cyanide into
the brain rather than the ratio of brain to blood 14CN activities
for several reasons. The determination of a meaningful ratio of
brain to blood concentrations requires that the distribution be at
equilibrium. However, cyanide is rapidly metabolized to thiocyanate by a ubiquitous enzyme, rhodanese, which is present
throughout the body, including the brain (Schulz, 1984; Way,
FIG. 3. The regional permeability-area products (regional PA) for cyanide
in the 10 brain structures according to treatment groups: (a) pons-medulla, (b)
cerebellum, (c) colliculi, (d) hypothalamus, (e) thalamus, (f) hippocampus, (g)
striatum, (h) frontal cortex, (i) parietal cortex, and (j) occipital cortex. The only
significant difference found was in the physiologic group where the PA of the
colliculi was significantly greater than in the hypothalamus (0.017 ⫾ 0.003,
0.009 ⫾ 0.001 s –1, respectively; p ⬍ 0.05).
ARTERIAL pH AND CYANIDE DISTRIBUTION
279
FIG. 5. The regional permeability-area products (regional PA) for cyanide in the pons-medulla, hypothalamus, thalamus, hippocampus, frontal, parietal, and
occipital cortices. The mean regional PAs of animals at acid pH were significantly greater than those of animals at alkaline pH for the following structures:
pons-medulla (p ⬍ 0.01), hypothalamus (p ⬍ 0.05), thalamus (p ⬍ 0.05), hippocampus (p ⬍ 0.05), frontal cortex (p ⬍ 0.05), parietal cortex (p ⬍ 0.05), and
occipital cortex (p ⬍ 0.05).
1984). In this context, the measurement of 14C radioactivity
does not permit the distinction of cyanide from its metabolite.
Thus, measurement of the permeability-area product during the
distribution phase permits the assessment of the effects of pH
while minimizing the effect of metabolism. In order to respect
the limits of Ohno’s method, we performed a preliminary
kinetic study demonstrating that blood and brain 14CN activities were measured during the phase of distribution.
Cyanide concentrations are usually measured in whole
blood. However, cyanide rapidly accumulates into erythrocytes
(McMillan and Svoboda, 1982; Vesey and Wilson, 1978) while
the plasma cyanide is the biochemically active fraction that
determines tissue levels and toxic effects (Vesey, 1979). There
is considerable evidence that cyanide toxicity correlates better
with plasma cyanide concentrations (Christel et al., 1977;
Marrs and Bright, 1987; Marrs et al., 1982, 1985). Thus, in the
distribution study, we studied plasma rather than whole blood
14
CN activity.
We found no significant effect of respiratory acidosis-alkalosis on the regional blood volumes as measured by 3H sucrose
activity. It should be noted that the regional blood volumes we
measured in animals in the physiologic condition were in the
same range as those previously reported by Ohno et al. (1978).
We found a low PA value at physiological pH for cyanide of
0.011 ⫾ 0.001 s –1. The in vivo kinetics of the penetration of
cyanide into the brain have been poorly investigated. In a
whole-body autoradiography study in mice, the central nervous
system had the lowest activity of all the tissues examined
(Clemedson et al., 1960). Postmortem findings showed that
cyanide concentrations in both white and gray matter of sheep
killed by an intramuscular injection of KCN were lower than
the blood concentrations. The mean concentration of cyanide
found in the brain was 27–30% of the mean concentration in
whole blood (Ballantyne, 1975). Furthermore, in fatal human
cases of acute cyanide poisoning, the brain concentrations were
consistently very much lower than the blood concentrations
280
DJERAD ET AL.
(Ballantyne and Marrs, 1987). In contrast, Oldendorff reported
a high olive oil partition coefficient and a high brain uptake
index for cyanide (Oldendorf, 1974). These conflicting results
require further studies to clarify the mechanisms and the in vivo
kinetics of the penetration of cyanide into the brain at both
nontoxic and toxic concentrations.
Our data show that respiratory acidification and alkalinization induce a significant effect on the rapidity of the distribution of intravenously administered cyanide into the brain. This
result was unexpected as the pK a of cyanide of 8.99 at 35°C
(Izatt et al., 1962) is well above the upper limit of pK a
considered to be significantly influenced by alteration of physiologic pH (Rowland and Tozer, 1995). Both respiratory acidosis (pH: 7.07 ⫾ 0.01) and alkalosis (pH: 7.58 ⫾ 0.01)
significantly modified the permeability-area product in 7 of the
10 studied brain structures.
The same dose of labeled cyanide was administered to
animals in the 3 treatment groups. Accordingly, there were no
significant differences of the 14CN activities measured in the
blood specimen collected at the neck after decapitation nor in
the arterial plasma specimens. In contrast, the PAs in animals
of the acidotic group were consistently greater than the PAs
measured in the alkalotic group. Thus, respiratory acidosis
increased the distribution of cyanide into rat brain in comparison with respiratory alkalosis. As the distribution of cyanide
into neuronal tissue was shown to be a nonenergy-dependent
process (Borowitz et al., 1994), the distribution of cyanide into
the brain depends on the effect of respiratory acidosis/alkalosis
on: (1) the binding of cyanide to plasma proteins, (2) the ratio
of nonionized to ionized forms of cyanide, and (3) the cerebral
blood flow (Goldberg et al., 1961). At micromolar concentrations, 60% of cyanide is bound to plasma proteins (Christel et
al., 1977). However, we used an extremely low dose of cyanide. Such a low dose may modify the binding of cyanide to
proteins and red blood cells, and consequently its tissue distribution pattern. Furthermore, we are not aware of any study
dealing with the effect of modulation of blood pH on the
binding of cyanide to plasma proteins. Unfortunately, we did
not measure the cerebral blood flow in our study. Numerous
factors may result in a modification of the cerebral blood flow
in our study; these include not only the effects of respiratory
acidosis/alkalosis (Hossmann and Blöink, 1981; Kety and
Schmidt, 1948), but also ketamine (Hougaard et al., 1974) and
PaO 2 (Kety and Schmidt, 1948). However, there was no significant difference of the PaO 2, and we used the same anesthetic agents in the 3 treatment groups. Respiratory acidosis
dramatically increases the cerebral blood flow in rats, while
respiratory alkalosis decreases it (Hossmann, 1990). The pK a
of cyanide is 8.99 at 35°C (Izatt et al., 1962). According to the
Henderson-Hasselbach equation, the nonionized to ionized ratio of cyanide would be 96.1 at a pH of 7.58, and 98.8 at a pH
of 7.07. These data suggest that respiratory acidosis/alkalosis
can significantly modify the nonionized to ionized ratio of
cyanide.
Our study suffers several limitations. We studied the distribution of cyanide into the brain at only 30 s after its administration. Thus, we have no insight regarding the duration of the
effect of the modulation of arterial pH on the brain distribution
of cyanide. Furthermore, we cannot clarify the brain structure(s) in which 14CN activity accumulates. In the study of
Clemedson et al. (1960), the central nervous system seemed to
have the lowest activity of all the tissues examined. This may
be due to a special process existing at the level of the bloodbrain barrier. Thus, 14CN activity measured in whole brain may
correspond to cyanide in the endothelial cells of the bloodbrain barrier and not to cyanide in the brain tissue. Other
methods such as brain microdialysis (Benveniste and Huttemeier, 1990) or capillary depletion (Triguero et al., 1990),
allowing access to the postcapillary compartment, would give
meaningful results regarding the effect of modulation of arterial pH on the distribution of cyanide into the brain.
The range of arterial pH used in this study may be encountered in acute human poisonings. There was a 47% reduction of
the permeability-area product of cyanide into the brain under
alkaline conditions compared with acidosis. This fact may be
of great importance in the clinical management of human
cyanide poisoning, especially in the setting of smoke inhalation, where inhalation of high concentrations of carbon dioxide
may cause acidosis and augment tissue distribution of cyanide.
Furthermore, our data suggest that respiratory alkalosis may be
protective with regard to the rapidity of cyanide distribution
into the brain. However, we studied a nontoxic dose of cyanide. Thus, definitive conclusions about the role of respiratory
acidosis/alkalosis in cyanide poisoning must await studies employing toxic doses.
In summary, the pharmacokinetics of a nontoxic dose of
cyanide administered intravenously to rats follows a 3-compartment model. The distribution is very rapid (whole blood
T 1/2␣ ⫽ 21.6 ⫾ 3.3 s) with an apparent volume of distribution
of 0.83 l/kg. The modulation of arterial blood pH by variation
of PaCO 2 significantly modifies the cerebral distribution of a
nontoxic dose of cyanide in rats. At a mean arterial pH of
7.58 ⫾ 0.01, the rapidity of cyanide distribution into the brain
assessed by the measurement of the permeability-area product
was 43% of that measured at a pH of 7.07 ⫾ 0.01. This effect
of pH on cyanide distribution does not appear to be limited to
specific areas of the brain, with 7 of 10 structures studied
showing significant differences between acidotic and alkalotic
conditions. Thus, modulation of arterial pH by altering PaCO 2
may induce significant effects on the brain uptake of cyanide.
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