Toxicology, 62 (1990) 213 --226 Elsevier Scientific Publishers Ireland Ltd.
Effects of ethanol in acute carbon monoxide poisoning Pawan Sharma and David G. Penney Department of Physiology, Wayne State University School of Medicine, Detroit, M1 48201 (U.S.A.) (Received September 1 lth, 1989; accepted December 26th, 1989)
Summary The combined effects of ethyl alcohol (ETOH) intoxication and carbon monoxide (CO) poisoning were studied in the Levine-prepared rat. Infusion or injection of ETOH before and during 90 min of CO exposure to blood levels 2--4 times those considered legally drunk in humans, increased survival at 2400 ppm, and extended the tolerance time at 2400 ppm and 3000 ppm. CO exposure produced the usual hypothermia, hypotension and hemoconcentration; these responses were not altered by concurrent ETOH treatment. Blood ETOH concentration was increased in the presence of CO, and this was related to CO concentration. Although ETOH did not alter the average degree of hypoglycemia seen during the later stages of CO exposure, rats with the highest ETOH concentration tended to have the lowest blood glucose. ETOH increased the magnitude of the hyperglycemic rebound during recovery from exposure to both CO concentrations. Moreover, the magnitude of the recovery hyperglycemic rebound was directly related to the magnitude of the previous hypoglycemia, at both CO concentrations, with or without ETOH. Rats dying during exposure to both CO concentrations were severely hypoglycemic, whereas survivors maintained more or less normal blood glucose concentrations. The results suggest that the presence of ETOH during CO poisoning increases blood ETOH to higher than expected levels and provides a significant degree of survival protection.
Key words: Brain damage; Carbon monoxide; Ethanol; Glucose; Hematocrit; Hypothermia; Morbidity; Mortality rate
Introduction C a r b o n m o n o x i d e is a c o l o r l e s s , o r d o r l e s s a n d n o n - i r r i t a t i n g g a s p r o d u c e d b y the incomplete combustion of carbon containing fossil fuels. Common sources of CO include motor vehicle exhaust, and malfunctioning heating and cooking e q u i p m e n t . A s t h e r e s u l t o f a c u t e p o i s o n i n g s , C O is a n n u a l l y r e s p o n s i b l e f o r 3 5 0 0 a c c i d e n t a l a n d s u i c i d a l d e a t h s i n t h e U S A [1]. It is a l s o a f r e q u e n t c a u s e o f s h o r t and long-term morbidity.
Address all correspondence to: David G. Penney, Ph.D., Associate Professor, Department of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201 (U.S.A.) 0300-483X/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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Approximately 90% of Americans drink ethyl alcohol (ETOH) at some time in their life, and 40--50% of men have problems with it [1]. Due to the fact that ETOH intoxication clouds judgement, can alter consciousness, and may impede escape from dangerous situations, opportunities abound for CO poisoning in individuals under its influence. ETOH was detected in 80% of adult fire victims in one study [2]. Those victims discovered in bed had a mean ETOH level of 268 mg/dl, whereas those near an exit had a mean level of 88 mg/dl. Few studies have examined the possible interactive effects of CO and ETOH. King [3] in a retrospective human investigation noted that the lethal CO level is higher in the presence of ETOH, suggesting that ETOH may provide some protection from CO. In contrast, Winston et al. [4] found that a 1 h pretreatment with ETOH increased mortality in mice exposed to 1900 ppm CO for 4 h. Because of its anesthetic effects which should lower metabolic rate and central nervous system (CNS) oxygen consumption, ETOH may provide protection from the "oxygen-starving" effect of high carboxyhemoglobin (COHb) saturation. ETOH's effect in producing peripheral vasodilation and lowering of the thermoregulatory set point [5] may also accelerate development of hypothermia in the CO poisoned subject. This could provide additional CNS protection by lowering oxygen uptake; hypothermia has been shown to be beneficial when it occurs during CO poisoning [6]. ETOH could also have specific metabolic effects which could be beneficial or deleterious. For example, ETOH may alter blood glucose concentration during CO poisoning; high glucose has been implicated in worsening neurologic outcome in recovery from global cerebral ischemia [7], spinal cord ischemia [8], cardiac arrest [9--11], and stroke [12,13]. Recent studies (Penney et al., unpublished data) suggest that both hyperglycemia and hypoglycemia increase neurologic deficit during acute CO poisoning in the rat, and a human retrospective study [14] shows an association between high blood glucose on admission to the hospital for CO poisoning and subsequent brain damage. Hypoglycemia, on the other hand, can lead to coma, brain damage, and if untreated, death [15,16]. Because ETOH can produce hypoglycemia [17], a fall in blood glucose could conceivably complicate CO poisoning. It was the goal of this study to examine in an animal model, the separate and combined effects of acute CO poisoning and ETOH intoxication on body temperature, heart rate, arterial blood pressure, hematocrit, blood glucose and ETOH concentration, neurologic deficit, and mortality. In particular, we wished to determine whether ETOH alters survival and neurologic outcome, and how it affects the glycemic pattern during and after CO exposure. The results suggest that ETOH provides modest survival protection during CO poisoning, attains higher blood levels in the presence of CO, may worsen hypoglycemia in some subjects and predispose to death, and exacerbates rebound hyperglycemia following CO exposure without altering neurologic outcome. Materials and methods
Animal preparation Female Sprague--Dawley rats (Charles River Breeding Labs) were used in the
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study. Two days prior to the experiment, a modified Levine preparation was produced [18,19]. The left c o m m o n carotid artery and jugular vein were cannulated with PE-50 polyethylene tubing under Ketamine (80 m g / k g ) and R o m p u n (4.8 m g / k g ) anesthesia, as previously described [19]. The cannulas were threaded under the skin to the nape of the neck, tied in place, and were plugged with Amphenol pins (No. 220-P02-100) when not in use. This procedure effectively ablates both vessels, placing one side of the brain at increased ischemic risk, and provides ports for infusion of agents and for monitoring of vital functions and for withdrawal of blood. The rats were returned to plastic shoebox cages and were housed individually. As earlier, they received food (Purina Rodent Chow) and water ad libitum. The animals were not fasted prior to CO exposure, although we did not monitor food intake.
Experimental design and CO exposure The rats were randomly assigned to one of five treatment groups as follows: (1) those to receive ethanol (4 g of 100% E T O H / k g body wt.) without CO, (2) those to inhale 2400 p p m CO without E T O H , (3) those to inhale 2400 p p m CO with E T O H (same dose as 1), (4) those to inhale 3000 p p m CO without E T O H , and (5) those to inhale 3000 p p m CO with E T O H (same dose as 1). They were exposed to 2400 p p m CO or 3000 p p m CO for 90 rain in the unanesthetized state. CO exposure took place in a large transparent plastic bag [19]. Surviving rats recovered in r o o m air for 24 h. R o o m temperature was 23--25 °C. The gas mixtures were made up by mixing air from a small compressor (40 1/ min) with CO from a commercial cylinder (Cryogenic Gases, Detroit), as needed. CO concentration was continuously monitored with a Beckman model 870, solid state, infrared analyzer, calibrated with a standard CO (2970 ppm)/nitrogen gas mixture. The carotid arterial blood pressure and heart rate were recorded on a Gould model 2400 pressurized-inking chart recorder. Small samples of blood were withdrawn f r o m the carotid cannula at intervals of 0, 45, 90, 210 and 330 min for the determination of hematocrit, blood glucose, and blood E T O H levels. The E T O H solution was made up by mixing equal quantities of 100% E T O H and distilled water. It was infused through the jugular cannula at a dose of 4 g E T O H / k g body weight. One-half the dose was given in a 10-min period immediately prior to CO exposure, and one-half was given during the 90 min of CO exposure. A H a r v a r d Co. infusion/withdrawal p u m p (model 940) was used for infusion. Rats were usually exposed in pairs, one receiving CO alone, the other CO and E T O H . The rats were confined in transparent lucite restrainers beginning 60 min before CO exposure began. This pre-CO exposure period allowed the rats to relax, providing more exact resting body temperatures.
Measurements Body temperature was monitored with a Yellow Springs Instrument Co. thermister probe inserted into the rectum. Hematocrit was determined by the standard microhematocrit method. Blood glucose was determined colorimetrically by a glucose oxidase method [20]. Blood ethanol was determined by an enzymatic method, a minor modification of the Sigma Chemical Co. procedure No. 332-
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UV. Optical density was recorded at 340 nm. E T O H concentration was calculated from the standard curve. Neurologic status, determined on a behavioral basis according to a system modified f r o m Lundy et al. [21], was assessed before and immediately after CO exposure (0 and 90 min), and after 2, 4 (210 and 330 min) and 24 h of r o o m air recovery. Scores of 1--5 were given in each of six areas: appearance, posture, shuffle (walking on front legs only), circling, activity, and screen (ability to hang onto a screen). Using this system, a neurologically normal rat received a score of six; whereas, a rat with severe neurologic deficit received a score of up to 30 [19]. Carboxyhemoglobin (COHb) saturation has recently been measured at the CO concentrations used here by an improved spectrophotometric method [22].
Data analysis The " E x c e l " spreadsheet and " C r i c k e t - G r a p h " programs running on a Macintosh SE microcomputer were used for data management, graphing and determination of inter-parameter correlation. Student's t-test and the method of analysis of variance were used for statistical analysis. Differences that resulted in P values of 0.05 or smaller were considered significant. Results
Acute severe CO poisoning in the rat results in hypothermia, hypotension, and hemoconcentration [19]. All three phenomena were seen in the present study, but few significant changes were related to the presence/absence of E T O H or to CO concentration (Table I). Body temperature fell 4 . 2 - - 4 . 6 ° C during CO exposure, with or without E T O H . Recovery of body temperature was retarded in the presence of E T O H at both CO concentrations, but the differences were not significant (data not shown). E T O H alone caused a 1.1 °C fall in body temperature. Mean arterial blood pressure fell 31--40 m m Hg during CO exposure, with or without E T O H . Recovery of blood pressure was significantly retarded by E T O H at 2400 p p m CO (CO alone, 82.4 _ 24.6°70; CO + E T O H , 3.3 _ 13.9070), but not at 3000 p p m CO. E T O H alone caused no change in blood pressure. Hematocrit increased 4.6--6.4070 during CO exposure, with or without E T O H . During recovery, hematocrit declined 5 . 0 - - 1 0 . 7 % ; the larger decrease at 3000 p p m than at 2400 p p m CO approached statistical significance. E T O H alone resulted in no change in hematocrit during the first 90 min, but there was a small decrease during the subsequent 4 h. CO exposure resulted in no consistent change in heart rate, with or without E T O H . E T O H alone did not change heart rate. C O H b was estimated to be 84070 and 88070 at 2400 p p m and 3000 p p m CO, respectively.
Blood ethanol concentration Figure 1 shows blood E T O H concentration in rats infused with E T O H , with and without CO. E T O H increased to 230 m g / d l after 45 min in the E T O H alone group. Blood E T O H had risen to 300 m g / d l by the termination of E T O H infusion (90 min), then fell progressively during recovery. Exposure to CO significantly increased the blood E T O H level at 45 min of CO exposure, and after 120
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TABLE I C H A N G E S IN B O D Y T E M P E R A T U R E , HEART RATE, MEAN ARTERIAL BLOOD PRESURE, A N D H E M A T O C R I T 1N R A T S E X P O S E D T O C A R B O N M O N O X I D E F O R 90 rain
Treatment
Change during 90 min of CO exposure Body temp.
Heart rate
Blood press.
Hematocrit
(°C)
(b.p.m.)
(mmHg)
(°70)
- 1.1 ± 0.1 (12)
11.7 ± 14.3 (12)
- 1.0 ± 4.5 (12)
0.2 ± 0.3 (11)
2400 ppm CO
- 4.5 ±0.2 (4)
3.3 ±49.1 (3)
- 31.3 ±2.9 (3)
4.6 ±2.0 (4)
2400 ppm CO ± ETOH
-- 4.2 ±0.3 (9)
7.8 ±22.0 (9)
- 38.2 ±5.1 (9)
5.3 ±0.6 (10)
3000 p p m CO
- 4.6 ±0.4 (7)
- 57.1 ±42.5 (7)
- 35.4 ± 8.8 (7)
6.4 ± 1.1 (7)
3000 p p m CO _ ETOH
-4.3 ±0.3 (10)
- 11.7 ± 16.4 (6)
-40.0 ±9.5 (7)
5.8 __.0.7 (7)
ETOH
alone
Values are means ± S . E . M . Number of animals used is given in parentheses.
and 240 min of recovery. Blood ETOH level was somewhat higher at 3000 ppm than at 2400 p p m at all four time points examined, reaching statistical significance after 90 min of CO exposure. Blood ETOH concentrations measured prior to CO exposure or ETOH infusion were close to zero, within the error of the assay method. ETOH was not measured in the rats treated with CO alone.
Blood glucose concentration Blood glucose concentration during and after CO exposure showed the characteristic "camel-back" response pattern previously observed (Penney et al., unpublished data) (Fig. 2). That is, blood glucose concentration was elevated after 45 min of CO exposure, fell to a normal or subnormal level by the termination of CO exposure, then rebounded after 2 h of recovery. Initial hyperglycemia was greater at 2400 ppm than at 3000 ppm CO, whether or not ETOH was present. Subsequent hypoglycemia was less at 2400 ppm than at 3000 ppm CO whether or not ETOH was present. Although the rate of decline of blood glucose was similar at both CO concentrations, the glucose concentration at 2400 ppm CO started at a higher level at 45 min, thus maintaining a higher level at 90 min. Plots of the change in glucose concentration 0--45 min versus the change in glu-
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500 (+)
"t-'#-
400 '
d
[]
ETOHAlone
[] []
2400 ppm CO + ETOH 3000 ppm CO + ETOH
+++
Z 0 0 300 ._1 0 Z < -1I-- 200 LLI
0.05). With 3000 p p m CO, 5 of 10 rats died both with and without E T O H . At both CO concentrations, those rats that died, did so at a later time when they had received E T O H than when E T O H was absent. In this regard, a Chi-square analysis of the
100 90
(1)
[~
80
LU I-< rr" 7O _J < _> SO > rr 5O
3000 CO (10) 3000 CO+ ETOH (10) 2400 CO (7) 2400 CO + ETOH (10)
~ ~ _ _
"
"
,,~"
f/
40
10
I
I
I
I
I
I
10
30
50
70
90
110
0
f /
i
I
210
i
I"
330
TIME (min) Fig. 3. Survial rate of rats exposed to carbon monoxide for 90 rain. Starting number of animals in
each group indicated in parentheses.
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combined 2400 p p m and 3000 p p m mortality data at 45 min of CO exposure indicated a statistically significant difference (df = 1, P < 0.01). None of the 12 rats given E T O H alone died. The E T O H treated rats, at both CO concentrations, that died had significantly lower blood glucose at the time of death than those which survived after 90 min of CO exposure [(died, 2400 ppm, 9.1 mg/dl; 3000 ppm, 16.3 _ 6.1 mg/dl) (lived, 2400 ppm, 81.0 ± 13.6 mg/dl; 3000 ppm, 91.3 ± 24.0 mg/dl)]. Tests of mortality versus hypoglycemia using Chi-square at 2400 p p m and 3000 p p m CO gave 4.44 (df = 1, P < 0.05) and 6.67 (df = 1, P < 0.01), respectively. Hypoglycemia in this instance was defined as glucose concentration < 40 m g / d l [23]. Blood glucose in the rats destined to die was also considerably lower than survivors after 45 min of CO exposure. This association of severe hypoglycemia and death during CO poisoning was noted earlier (Penney et al., unpublished data). At both CO concentrations blood E T O H concentration tended to be higher in non-survivors at the time of death than after 90 min of CO exposure in survivors, but the differences were not significant. Moreover, those rats with the highest E T O H concentration tended to have the lowest blood glucose concentration (r = 0.51, P < 0.05). A m o n g the 3000 p p m rats treated with E T O H , non-survivors showed a much larger increase in hematocrit during the first 45 min of CO exposure (6.7 ± 0.1%0 vs. 3.6 ___ 0.6%).
Neurologic index Rats given E T O H alone displayed a neurologic index of 17.8 at the termination of E T O H infusion (90 min), reflecting their intoxicated condition (Fig. 4). Neurologic index then fell progressively until, by 24 h it was not differ-
[]
+++ (+) + ++
26 X HJ a Z
ETOH Alone
[]
2400 ppm CO Alone
[]
2400 ppm CO + ETOH
i
3000 ppm CO Alone
[]
3000 ppm CO + ETOH
o
16
w Z
12
10
Z
9
12 iii~4
i. 120
9O
PERIOD
24 hrs
240
OF CO EXPOSURE AND RECOVERY
(min)
Fig. 4. Neurologic index of rats following carbon monoxide exposure. Compared to the ethanol alone group: *, P < 0.05; *+, P < 0.01; ++*, P < 0.001. Compared to the 2400 ppm CO alone group: ( + ) , 1 S.E.M. Number of animals indicated at the base of columns.
220
ent from 6 (i.e. normal), suggesting that blood E T O H was near zero. Combined CO exposure and E T O H treatment increased neurologic index above that with E T O H alone, and with 2400 p p m CO after 90 min of exposure and after 120 and 240 min of recovery the increase nearly equalled the effect of E T O H alone at those times. With 3000 p p m CO, the increase in neurologic index at 120 and 240 min was somewhat greater than the effect of E T O H alone at those times. After 24 h of recovery, there was no significant difference in neurologic index between 3000 p p m CO rats receiving E T O H and those not receiving E T O H , suggesting that E T O H exerts neither a protective nor a deleterious effect on brain function at this CO concentration. Neurologic index was not determined at 24 h for rats exposed to 2400 p p m CO alone. Neurologic index was significantly higher at 240 min of recovery in rats exposed to 3000 versus 2400 p p m CO, indicating a doseresponse relationship between CO concentration and neurologic deficit. It should be noted that when deaths occurred, rats with the greatest potential neurologic deficit were eliminated, thus the neurologic index values from groups with high mortality are biased on the low side. Also, the regression plots do not contain data on rats which died during CO exposure, the ones which incurred the most severe hypoglycemia. Discussion Consistent with past reports of acute CO poisoning [19], we observed hypothermia, hypotension and evidence of hemoconcentration in the CO-exposed rats, which was not altered by E T O H treatment. The blood E T O H concentration attained in the rats was 2 - - 4 times the human legal intoxication level (100 m g / dl). However, it was well below the lethal blood level of 890--1000 m g / d l for rats receiving E T O H alone [24]. Whether the lethal E T O H level in the presence of CO is lower, is unknown. While not markedly altering the neurologic deficit obtained 24 h after CO poisoning, a time when neither CO nor E T O H were present in the animals, E T O H appeared to increase the survival rate at 2400 p p m CO and the tolerance time at both CO concentrations. If CO exposure had stopped before the 90 min m a r k (e.g. 45 min), the differences in survival rate between rats given E T O H and those not given E T O H would have been larger. Not only was blood E T O H concentration increased in the presence of CO, but the increase was related to CO concentration. E T O H exerted no effect on the average degree of hypoglycemia which developed late in CO poisoning, but the hypoglycemia was worsened by higher CO concentration. In contrast, the hyperglycemic rebound following CO exposure was increased by E T O H . Our results also show that early hyperglycemia was protective of later severe hypoglycemia, at both CO concentrations, whether or not E T O H was present. During recovery, the magnitude of the hyperglycemic rebound was directly related to the magnitude of the previous hypoglycemia, again at both CO concentrations, with or without E T O H . An examination of the data for the survivors and the non-survivors revealed severe hypoglycemia in the latter, and more or less normal blood glucose in the former, whether or not E T O H was present. The association of severe hypoglycemia and death has been noted earlier (Penney et al., unpublished
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data). Because the rats with the highest blood E T O H tended to be non-survivors, and those rats also tended to have lower blood glucose, E T O H would appear to be disadvantageous to survival in this regard, in some subjects. As is well known, CO exerts its toxic effects by avidly binding to hemoglobin. In so doing, it renders a fraction o f the hemoglobin incapable of transporting oxygen and decreases the ease of tissue oxygen unloading. Mainly due to the latter effect, an hypoxic state exists which is more severe than that of an hypoxic hypoxia of similar arterial oxygen saturation. Whereas previous studies described only a hyperglycemic response to CO poisoning [25,26], recent experiments suggest that the response is more complex. Hyperglycemia develops during early CO exposure, but is then attenuated or reversed during late CO exposure; a second hyperglycemic surge occurs during early recovery, followed by a return to normal values. We have termed this a "camel-back" pattern. Hypoglycemia is exacerbated by increased CO concentration, saline administration, and the prevention of CO-induced hypothermia (Sutariya et al., unpubl, data). Increased neurologic deficit has been correlated with a larger post-CO hyperglycemic rebound (Penney et al., unpublished data). Approximately 90--98% of ingested E T O H is oxidized by the liver, while the remainder is excreted unchanged [27]. As with other drugs [28], the rate of E T O H metabolism is dependent upon hepatic blood flow. Sylvester et al. [29] has shown that for a given level of arterial oxygen saturation, hypoxic hypoxia results in a relatively greater shunting of blood from the splanchnic vascular bed to the liver than does CO hypoxia. This could explain the higher blood E T O H concentration we observed in the CO-exposed rats. Topping et al. [30] report that 20% COHb saturation decreases E T O H utilization in the in vitro liver by one-half, and also decreases E T O H uptake. On the other hand, the hemoconcentration during CO exposure and the hemodilution following CO exposure, which we have inferred on the basis o f changes in hematocrit, should not have affected the plasma E T O H concentration, because E T O H is fairly uniformily distributed throughout all tissues and all fluids of the body [27]. CO-induced hemoconcentration was probably due to a redistribution of body fluids. Ogawa et al. [31] observed a 20% fall in blood volume after 30 min at 60% COHb saturation, mainly resulting from a sharp reduction in plasma volume caused by increased vascular permeability. Pohorecky and Newman [32] found that E T O H in doses of 1, 2 and 4 g/kg produced 4.7%, 7.3% and 15.3% increases in hematocrit, respectively, after 1 h. The highest E T O H dose decreased plasma volume by 20%, indicating that E T O H induces fluid loss from the vascular compartment. Nonetheless, we saw no evidence of hemoconcentration in our rats given E T O H alone, and no additional hemoconcentration occurred in the CO-exposed rats treated with E T O H . It is significant that E T O H treated rats which died during CO-exposure experienced greater increases in hematocrit than the survivors. This suggests that the vascular damage in the non-survivors was greater, but whether this was due to the hypoglycemia per se, is unknown. E T O H and other alcohols are reported to provide protection during hypoxic hypoxia [33,34]. This protective effect of E T O H could involve several different mechanisms: (1) an anesthetic depressant action lowering CNS metabolic rate
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[27], (2) an induction of hypothermia also reducing CNS metabolic rate, and (3) a specific metabolic action. A reduction in metabolic rate by whatever means would decrease the need for oxygen and prevent damage to sensitive aerobic organs, such as the brain. Sutariya et al. [6] showed that CO-induced hypothermia, or hypothermia produced by pre-cooling, improves survival and neurologic outcome in CO-poisoned rats. Lomax et al. [5] reported that ETOH-(0.5--3.2 g/ kg body wt) causes a dose-dependent fall in body temperature in rats. A 1.5 g/kg dose caused a fall of 1.6°C over 60 min at an environmental temperature of 18°C, not too different from the 1.1 °C decrease over 90 min observed in the present study at a 25°C room temperature. However, E T O H administration in our CO-exposed rats produced no greater hypothermia than in rats given CO alone, suggesting that CO's interference with body temperature control is much greater than that of E T O H , at least at the CO concentrations used in the present study. Some investigators [33] suggest that E T O H may provide benefit through development of ketosis, which by oxidizing ketones for energy production, minimizes the deleterious accumulation of brain lactic acid, since development of acidosis is postulated to play a major role in cell damage [35]. In this regard, significant increases in beta-hydroxybutyrate and glycerol have been reported in pigs infused with E T O H [36]. Low blood glucose and ketosis in fasted rats has been suggested to explain an increased tolerance of extreme hypoxia, as compared to fed rats [37]. E T O H is reported to decrease [17], increase [38], and not change [39] blood glucose in fed rats; however, we saw no hypoglycemia in our non-fasted rats given E T O H , and E T O H failed to exacerbate the hypoglycemic action of CO. Marks [40], in a review, suggests that E T O H may contribute to the development of hypoglycemia in two ways: (1) indirectly through its effects on hypothalamicpituitary-adrenal function, and (2) directly by impairing hepatic gluconeogenesis. Arky [41] suggests that gluconeogenesis is inhibited by E T O H due to the increase in the N A D H / N A D ratio, which inhibits the entrance of glycerol, lactate, and specific amino acids into metabolic pathways by which these metabolites are converted to glucose. CO hypoxia may do something similar. Hyperglycemic rebound with or without E T O H may occur for two reasons: (1) reversal of CO-hypoxia which occurs in rats within 2 h after transfer to room air [28], thus unblocking gluconeogenesis for the reasons described above, and (2) CO-stimulated release of catecholamines [42] acting on the liver to increase glycogenolysis and glucose release. In addition to these factors, insulin release is suppressed and glucagon and corticosteroid release is stimulated by E T O H [17,35,43]. Plasma insulin is also lowered by CO exposure (Penney et al., unpublished data). High blood E T O H concentration during recovery may have continued to perturb the levels of these hormones as indicated, providing an environment for development of greater hyperglycemia. Because catecholamine release is also stimulated by E T O H [44], E T O H could magnify the hyperglycemic response. However, we did not measure any of these hormones in our study. Other factors may also be involved. As observed in this experimental model the effects of CO are far more prominent than those of E T O H in terms of heart rate, blood pressure, hematocrit, and
223
b l o o d g l u c o s e , at least at t h e d o s e levels o f C O a n d E T O H u s e d . E T O H a p p e a r s to p r o v i d e s i g n i f i c a n t s u r v i v a l p r o t e c t i o n f r o m C O p o i s o n i n g ; a s h o r t e r p e r i o d o f C O e x p o s u r e at t h e C O c o n c e n t r a t i o n s u s e d h e r e w o u l d h a v e p r o d u c e d n e a r l y 1 0 0 % s u r v i v a l in E T O H t r e a t e d a n i m a l s . S i n c e E T O H levels a r e e l e v a t e d in t h e presence of CO, might an individual ingesting ETOH, more rapidly reach the l e t h a l level in t h e p r e s e n c e o f C O ? O n t h e o t h e r h a n d , t h e p r e s e n c e o f E T O H d o e s n o t a l t e r t h e d e g r e e o r r a p i d i t y o f d e v e l o p m e n t o f h y p o g l y c e m i a , w h i c h is associated with death. However, ETOH does increase the hyperglycemic rebound, a l t h o u g h it d o e s n o t a p p e a r t o i n c r e a s e n e u r o l o g i c d e f i c i t d u r i n g r e c o v e r y .
Acknowledgements T h i s w o r k was s u p p o r t e d b y a r e s e a r c h g r a n t f r o m t h e A m e r i c a n H e a r t A s s o c i a t i o n o f M i c h i g a n . W e w i s h to t h a n k L i n d a M c C r a w f o r h e l p in p r e p a r a t i o n o f the manuscript.
References 1
2 3 4
5 6 7 8
9 10 11
12 13 14 15
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E. Braunwald, K.J. Isselbacher, R.G. Petersdorf, J.D. Wilson, J.B. Martin and A.S. Fauci, Harrison's Principles of Internal Medicine, llth Ed., McGraw-Hill Book Co., N.Y., 1987, pp. 843 and 2106. D.J. BariIlo, B.F. Rush, R. Goode, R.-L. Lin, A. Freda and E.J. Anderson, Is ethanol the unknown toxin in smoke inhalation injury. Amer. Surgeon, 52 (1986) 641. L.A. King, Effect of ethanol in fatal carbon monoxide poisonings. Hum. Toxicol., 2 (1983) 155. J.M. Winston, J.M. Creighton and R.J. Roberts, Alteration of carbon monoxide and hypoxic hypoxia-induced lethality following phenobarbital, chlorpromazine, or alcohol pretreatment. Toxicol. Appl. Pharmacol., 30 (1974) 458. P. Lomax, J.G. Bajorek, W.A. Chesarek and R.R.J. Chaffee, Ethanol induced hypothermia in the rat. Pharmacology, 21 (1980) 228. B. Sutariya, D. Penney, J. Barnes and C. Helfrnan, Hypothermia protects brain function in acute carbon monoxide poisoning. Vet. Hum. Toxicol., 31 (1989) 436. K. Siemkowicz and A.J. Hansen, Clinical restitution following cerebral ischemia in hypo-, normo- and hyperglycemic rats. Acta Neurol. Scand., 58 (1978) 1. D.R. LeMay, S. Neal, G.B. Zelenock and L.G. D'Alecy, Paraplegia in the rat induced by aortic cross-clamping: Model characterization and glucose exacerbation of neurologic deficit. J. Vasc. Surg., 6 (1987) 383. R.E. Myers and S.-I. Yamaguchi, Nervous system effects of cardiac arrest in monkeys. Arch. Neurol., 34 (1977) 65. W.T. Longstreth, Jr. and T.S. Inui, High blood glucose level on hospital admission and poor neurological recovery after cardiac arrest. Ann. Neurol., 15 (1984) 59. E.F. Lundy, J.E. Kuhn, J.W. Kwon, G.B. Zelenock and L.G. D'Alecy, Infusion of five percent dextrose increases mortality and morbidity following six minutes of cardiac arrest in resuscitated dogs. J. Crit. Care, 2 (1987) 4. M.C. Riddle and J. Hart, Hyperglycemia, recognized and unrecognized, as a risk factor for stroke and transient ischemic attacks. Stroke, 13 (1982) 356. C.M. Helgason, Blood glucose and stroke. Current concepts of cerebrovascular disease and stroke, 23 (1988) 1. D.G. Penney, Hyperglycemia exacerbates brain damage in acute severe carbon monoxide poisoning. Med. Hypotheses, 27 (1988) 241. L.L. Madison, Ethanol-induced hypoglycemia. In R. Levine, R. Luft (Eds.), Adv. Metab. Dis., Vol. 3, Academic Press, NY, 1968, p. 85.
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26 27 28 29 30 31 32 33 34 35 36 37 38
39 40 41
R.N. Auer, Progress Review: Hypoglycemic brain damage. Stroke, 17 (1986) 699. D.C. Holley, G.J. Ragby and D.L. Curry, Ethanol-insulin interrelationships in the rat studied in vitro and in vivo; evidence for direct-ethanol inhibition of biphasic glucose-induced insulin secretion. Metabolism, 30 (1981) 894. S. Levine, Anoxic ischemic encephalopathy in rats. Am. J. Pathol., 36 (1960) 1. D.G. Penney, K. Verma and J.A. Hull, Cardiovascular, metabolic and neurologic effects of acute carbon monoxide poisoning in the rat. Toxicol. Letts., 45 (1989) 207. A.St.G. Huggett and D.A. Nixon, Use of glucose oxidase, peroxidase and o-dianisidine in determination of blood and urinary glucose. Lancet (1957) 368. E.F. Lundy, J. Dykstra, B. Luyckx, G.B. Zelenock and L.G. D'Alecy, Reduction of neurologic deficit by 1,3-butanediol induced ketosis in Levine rats. Stroke, 16 (1985) 855. K. Verma, D.G. Penney, C.C. Helfman and B.B. Sutariya, Carboxyhemoglobin in the rat: Improvements in the spectrophotometric measurement and comparison to other studies. J. Appl. Toxicol., 9 (1989) 323. E.C. Klatt, C. Beatie and T.T. Noguchi, Evaluation of death from hypoglycemia. Am. J. Forensic Med. Pathol., 9 (1988) 122. H.W. Haggard, L.A. Greenberg and N. Rakieten, Studies on the absorption, distribution and elimination of alcohol, VI. The principles governing the concentration of alcohol in the blood and the concentration causing respiratory failure. J. Pharmacol. Exp. Ther., 69 (1940) 252. M. Gothert, P. Hock, G. Malorny and H.-J. Seiler, Mechanisms of hyperglycemia during acute carbon monoxide poisoning. Naunyn-Schmiedeberg's Archiv. Pharmacol., 274 (Suppl. R41) (1972). D. Pankow and W. Ponsold, Uber das Verhalten des Blutglucospiegels nach Kohlenmonoxidvergiftung bei Ratten. Int. Arch. Arbeitsmed., 30 (1972) 210. L.S. Goodman and A. Gilman, The Pharmacological Basis of Therapeutics, 5th edn., Macmillan Publ., N.Y., 1975, p. 142. M.R. Montgomery and R.J. Rubin, The effect of carbon monoxide inhalation in vivo drug metabolism in the rat. J. Pharmacol. Exp. Ther., 179 (1971) 465. J.T. Sylvester, R.D. Gilbert and S. Permutt, Effects of hypoxic and carbon monoxide hypoxia on venous returns in dogs. Fed. Proc., 32 (1973) 395 (Abstr.). D.L. Topping, R.C. Fishlock, R.P. Trimble, G.B. Storer and A.M. Snoswell, Carboxyhemoglobin inhibits the metabolism of ethanol by perfused rat liver. Biochem. Int., 3 (1981) 157. M. Ogawa, S. Shimazaki, N. Tanaka, T. Ukai and T. Sugimoto, Blood volume changes in experimental carbon monoxide poisoning. Ind. Health, 12 (1974) 121. L.A. Pohorecky and B. Newman, Alterations in the vascular compartments with acute ethanol treatment. J. Pharm. Pharmacol., 30 (1978) 785. M.M. Moursi, B.A. Luyckx and L.G. D'Alecy, The role of ethanol in diluents of drugs that protect mice from hypoxia. Stroke, 14 (1983) 791. E.F. Lundy, B.A. Luyckx, D.J. Combs, G.B. Zelenock and L.G. D'Alecy, Butanediol induced cerebral protection from ischemic-hypoxia in the instrumented Levine rat. Stroke, 15 (1984) 547. S. Rehncrona, I. Rosen and B.K. Siesjo, Excessive cellular acidosis: An important mechanism of neuronal damage in the brain? Acta Physiol. Scand., 110 (1980) 435. A. Tiengo, M. Molinari, E. Marchiori, P. Frasson, D. Fedele, E. Ancona, G. Enzi and G. Crepaldi, Metabolic and hormonal effects of ethanol. Diabete Metab,, 4 (1978) 19. W.S. Myles, Survival of fasted rats exposed to altitude. Can. J. Physiol. Pharmacol., 54 (1976) 883. M.L. Oliviera-de-Souza and J. Masur, Blood glucose and body temperature alterations induced by ethanol in rats submitted to different levels of food deprivation. Pharmacol. Behav., 15 (1981) 551. J.L. Tramill, P.E. Turner, G. Harwell and S.F. Davis, Alcoholic hypoglycemia as a result of acute challenges of ethanol. Physiol. Psychol., 9 (1981) 114. V. Marks, Alcohol-induced hypoglycemia and endocrinopathy. In G. Edwards and M. Grants (Eds.), Alcoholism: New Knowledge and New Responses, London, Croom Helm, 1977, p. 208. R.A. Arky, E. Veverbrants and E.A. Abramson, Irreversible hypoglycemia. JAMA, 206 (1968) 575.
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J.T. Sylvester, S.M. Scharf, R.D. Gilbert and R.S. Fitzgerald, Hypoxia and CO hypoxia in dogs: Hemodynamics, carotid reflexes, and catecholamines. Am. J. Physiol., 236 (1979) H22. F.M. Sturtevant and R.P. Sturtevant, Ethanol and blood corticosteroid levels in chronobiology. Int. J. Pharmacol. Ther. Toxicol., 19 (1981) 427. R.H. Ylikahri, M.O. Huttunen and M. Harkonen, Hormonal changes during alcohol intoxication and withdrawal. Pharmacol. Biochem. Behav., 13 (Suppl. i) (1980) 131.
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Effects of ethanol in acute carbon monoxide poisoning Pawan Sharma and David G. Penney Department of Physiology, Wayne State University School of Medicine, Detroit, M1 48201 (U.S.A.) (Received September 1 lth, 1989; accepted December 26th, 1989)
Summary The combined effects of ethyl alcohol (ETOH) intoxication and carbon monoxide (CO) poisoning were studied in the Levine-prepared rat. Infusion or injection of ETOH before and during 90 min of CO exposure to blood levels 2--4 times those considered legally drunk in humans, increased survival at 2400 ppm, and extended the tolerance time at 2400 ppm and 3000 ppm. CO exposure produced the usual hypothermia, hypotension and hemoconcentration; these responses were not altered by concurrent ETOH treatment. Blood ETOH concentration was increased in the presence of CO, and this was related to CO concentration. Although ETOH did not alter the average degree of hypoglycemia seen during the later stages of CO exposure, rats with the highest ETOH concentration tended to have the lowest blood glucose. ETOH increased the magnitude of the hyperglycemic rebound during recovery from exposure to both CO concentrations. Moreover, the magnitude of the recovery hyperglycemic rebound was directly related to the magnitude of the previous hypoglycemia, at both CO concentrations, with or without ETOH. Rats dying during exposure to both CO concentrations were severely hypoglycemic, whereas survivors maintained more or less normal blood glucose concentrations. The results suggest that the presence of ETOH during CO poisoning increases blood ETOH to higher than expected levels and provides a significant degree of survival protection.
Key words: Brain damage; Carbon monoxide; Ethanol; Glucose; Hematocrit; Hypothermia; Morbidity; Mortality rate
Introduction C a r b o n m o n o x i d e is a c o l o r l e s s , o r d o r l e s s a n d n o n - i r r i t a t i n g g a s p r o d u c e d b y the incomplete combustion of carbon containing fossil fuels. Common sources of CO include motor vehicle exhaust, and malfunctioning heating and cooking e q u i p m e n t . A s t h e r e s u l t o f a c u t e p o i s o n i n g s , C O is a n n u a l l y r e s p o n s i b l e f o r 3 5 0 0 a c c i d e n t a l a n d s u i c i d a l d e a t h s i n t h e U S A [1]. It is a l s o a f r e q u e n t c a u s e o f s h o r t and long-term morbidity.
Address all correspondence to: David G. Penney, Ph.D., Associate Professor, Department of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201 (U.S.A.) 0300-483X/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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Approximately 90% of Americans drink ethyl alcohol (ETOH) at some time in their life, and 40--50% of men have problems with it [1]. Due to the fact that ETOH intoxication clouds judgement, can alter consciousness, and may impede escape from dangerous situations, opportunities abound for CO poisoning in individuals under its influence. ETOH was detected in 80% of adult fire victims in one study [2]. Those victims discovered in bed had a mean ETOH level of 268 mg/dl, whereas those near an exit had a mean level of 88 mg/dl. Few studies have examined the possible interactive effects of CO and ETOH. King [3] in a retrospective human investigation noted that the lethal CO level is higher in the presence of ETOH, suggesting that ETOH may provide some protection from CO. In contrast, Winston et al. [4] found that a 1 h pretreatment with ETOH increased mortality in mice exposed to 1900 ppm CO for 4 h. Because of its anesthetic effects which should lower metabolic rate and central nervous system (CNS) oxygen consumption, ETOH may provide protection from the "oxygen-starving" effect of high carboxyhemoglobin (COHb) saturation. ETOH's effect in producing peripheral vasodilation and lowering of the thermoregulatory set point [5] may also accelerate development of hypothermia in the CO poisoned subject. This could provide additional CNS protection by lowering oxygen uptake; hypothermia has been shown to be beneficial when it occurs during CO poisoning [6]. ETOH could also have specific metabolic effects which could be beneficial or deleterious. For example, ETOH may alter blood glucose concentration during CO poisoning; high glucose has been implicated in worsening neurologic outcome in recovery from global cerebral ischemia [7], spinal cord ischemia [8], cardiac arrest [9--11], and stroke [12,13]. Recent studies (Penney et al., unpublished data) suggest that both hyperglycemia and hypoglycemia increase neurologic deficit during acute CO poisoning in the rat, and a human retrospective study [14] shows an association between high blood glucose on admission to the hospital for CO poisoning and subsequent brain damage. Hypoglycemia, on the other hand, can lead to coma, brain damage, and if untreated, death [15,16]. Because ETOH can produce hypoglycemia [17], a fall in blood glucose could conceivably complicate CO poisoning. It was the goal of this study to examine in an animal model, the separate and combined effects of acute CO poisoning and ETOH intoxication on body temperature, heart rate, arterial blood pressure, hematocrit, blood glucose and ETOH concentration, neurologic deficit, and mortality. In particular, we wished to determine whether ETOH alters survival and neurologic outcome, and how it affects the glycemic pattern during and after CO exposure. The results suggest that ETOH provides modest survival protection during CO poisoning, attains higher blood levels in the presence of CO, may worsen hypoglycemia in some subjects and predispose to death, and exacerbates rebound hyperglycemia following CO exposure without altering neurologic outcome. Materials and methods
Animal preparation Female Sprague--Dawley rats (Charles River Breeding Labs) were used in the
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study. Two days prior to the experiment, a modified Levine preparation was produced [18,19]. The left c o m m o n carotid artery and jugular vein were cannulated with PE-50 polyethylene tubing under Ketamine (80 m g / k g ) and R o m p u n (4.8 m g / k g ) anesthesia, as previously described [19]. The cannulas were threaded under the skin to the nape of the neck, tied in place, and were plugged with Amphenol pins (No. 220-P02-100) when not in use. This procedure effectively ablates both vessels, placing one side of the brain at increased ischemic risk, and provides ports for infusion of agents and for monitoring of vital functions and for withdrawal of blood. The rats were returned to plastic shoebox cages and were housed individually. As earlier, they received food (Purina Rodent Chow) and water ad libitum. The animals were not fasted prior to CO exposure, although we did not monitor food intake.
Experimental design and CO exposure The rats were randomly assigned to one of five treatment groups as follows: (1) those to receive ethanol (4 g of 100% E T O H / k g body wt.) without CO, (2) those to inhale 2400 p p m CO without E T O H , (3) those to inhale 2400 p p m CO with E T O H (same dose as 1), (4) those to inhale 3000 p p m CO without E T O H , and (5) those to inhale 3000 p p m CO with E T O H (same dose as 1). They were exposed to 2400 p p m CO or 3000 p p m CO for 90 rain in the unanesthetized state. CO exposure took place in a large transparent plastic bag [19]. Surviving rats recovered in r o o m air for 24 h. R o o m temperature was 23--25 °C. The gas mixtures were made up by mixing air from a small compressor (40 1/ min) with CO from a commercial cylinder (Cryogenic Gases, Detroit), as needed. CO concentration was continuously monitored with a Beckman model 870, solid state, infrared analyzer, calibrated with a standard CO (2970 ppm)/nitrogen gas mixture. The carotid arterial blood pressure and heart rate were recorded on a Gould model 2400 pressurized-inking chart recorder. Small samples of blood were withdrawn f r o m the carotid cannula at intervals of 0, 45, 90, 210 and 330 min for the determination of hematocrit, blood glucose, and blood E T O H levels. The E T O H solution was made up by mixing equal quantities of 100% E T O H and distilled water. It was infused through the jugular cannula at a dose of 4 g E T O H / k g body weight. One-half the dose was given in a 10-min period immediately prior to CO exposure, and one-half was given during the 90 min of CO exposure. A H a r v a r d Co. infusion/withdrawal p u m p (model 940) was used for infusion. Rats were usually exposed in pairs, one receiving CO alone, the other CO and E T O H . The rats were confined in transparent lucite restrainers beginning 60 min before CO exposure began. This pre-CO exposure period allowed the rats to relax, providing more exact resting body temperatures.
Measurements Body temperature was monitored with a Yellow Springs Instrument Co. thermister probe inserted into the rectum. Hematocrit was determined by the standard microhematocrit method. Blood glucose was determined colorimetrically by a glucose oxidase method [20]. Blood ethanol was determined by an enzymatic method, a minor modification of the Sigma Chemical Co. procedure No. 332-
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UV. Optical density was recorded at 340 nm. E T O H concentration was calculated from the standard curve. Neurologic status, determined on a behavioral basis according to a system modified f r o m Lundy et al. [21], was assessed before and immediately after CO exposure (0 and 90 min), and after 2, 4 (210 and 330 min) and 24 h of r o o m air recovery. Scores of 1--5 were given in each of six areas: appearance, posture, shuffle (walking on front legs only), circling, activity, and screen (ability to hang onto a screen). Using this system, a neurologically normal rat received a score of six; whereas, a rat with severe neurologic deficit received a score of up to 30 [19]. Carboxyhemoglobin (COHb) saturation has recently been measured at the CO concentrations used here by an improved spectrophotometric method [22].
Data analysis The " E x c e l " spreadsheet and " C r i c k e t - G r a p h " programs running on a Macintosh SE microcomputer were used for data management, graphing and determination of inter-parameter correlation. Student's t-test and the method of analysis of variance were used for statistical analysis. Differences that resulted in P values of 0.05 or smaller were considered significant. Results
Acute severe CO poisoning in the rat results in hypothermia, hypotension, and hemoconcentration [19]. All three phenomena were seen in the present study, but few significant changes were related to the presence/absence of E T O H or to CO concentration (Table I). Body temperature fell 4 . 2 - - 4 . 6 ° C during CO exposure, with or without E T O H . Recovery of body temperature was retarded in the presence of E T O H at both CO concentrations, but the differences were not significant (data not shown). E T O H alone caused a 1.1 °C fall in body temperature. Mean arterial blood pressure fell 31--40 m m Hg during CO exposure, with or without E T O H . Recovery of blood pressure was significantly retarded by E T O H at 2400 p p m CO (CO alone, 82.4 _ 24.6°70; CO + E T O H , 3.3 _ 13.9070), but not at 3000 p p m CO. E T O H alone caused no change in blood pressure. Hematocrit increased 4.6--6.4070 during CO exposure, with or without E T O H . During recovery, hematocrit declined 5 . 0 - - 1 0 . 7 % ; the larger decrease at 3000 p p m than at 2400 p p m CO approached statistical significance. E T O H alone resulted in no change in hematocrit during the first 90 min, but there was a small decrease during the subsequent 4 h. CO exposure resulted in no consistent change in heart rate, with or without E T O H . E T O H alone did not change heart rate. C O H b was estimated to be 84070 and 88070 at 2400 p p m and 3000 p p m CO, respectively.
Blood ethanol concentration Figure 1 shows blood E T O H concentration in rats infused with E T O H , with and without CO. E T O H increased to 230 m g / d l after 45 min in the E T O H alone group. Blood E T O H had risen to 300 m g / d l by the termination of E T O H infusion (90 min), then fell progressively during recovery. Exposure to CO significantly increased the blood E T O H level at 45 min of CO exposure, and after 120
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TABLE I C H A N G E S IN B O D Y T E M P E R A T U R E , HEART RATE, MEAN ARTERIAL BLOOD PRESURE, A N D H E M A T O C R I T 1N R A T S E X P O S E D T O C A R B O N M O N O X I D E F O R 90 rain
Treatment
Change during 90 min of CO exposure Body temp.
Heart rate
Blood press.
Hematocrit
(°C)
(b.p.m.)
(mmHg)
(°70)
- 1.1 ± 0.1 (12)
11.7 ± 14.3 (12)
- 1.0 ± 4.5 (12)
0.2 ± 0.3 (11)
2400 ppm CO
- 4.5 ±0.2 (4)
3.3 ±49.1 (3)
- 31.3 ±2.9 (3)
4.6 ±2.0 (4)
2400 ppm CO ± ETOH
-- 4.2 ±0.3 (9)
7.8 ±22.0 (9)
- 38.2 ±5.1 (9)
5.3 ±0.6 (10)
3000 p p m CO
- 4.6 ±0.4 (7)
- 57.1 ±42.5 (7)
- 35.4 ± 8.8 (7)
6.4 ± 1.1 (7)
3000 p p m CO _ ETOH
-4.3 ±0.3 (10)
- 11.7 ± 16.4 (6)
-40.0 ±9.5 (7)
5.8 __.0.7 (7)
ETOH
alone
Values are means ± S . E . M . Number of animals used is given in parentheses.
and 240 min of recovery. Blood ETOH level was somewhat higher at 3000 ppm than at 2400 p p m at all four time points examined, reaching statistical significance after 90 min of CO exposure. Blood ETOH concentrations measured prior to CO exposure or ETOH infusion were close to zero, within the error of the assay method. ETOH was not measured in the rats treated with CO alone.
Blood glucose concentration Blood glucose concentration during and after CO exposure showed the characteristic "camel-back" response pattern previously observed (Penney et al., unpublished data) (Fig. 2). That is, blood glucose concentration was elevated after 45 min of CO exposure, fell to a normal or subnormal level by the termination of CO exposure, then rebounded after 2 h of recovery. Initial hyperglycemia was greater at 2400 ppm than at 3000 ppm CO, whether or not ETOH was present. Subsequent hypoglycemia was less at 2400 ppm than at 3000 ppm CO whether or not ETOH was present. Although the rate of decline of blood glucose was similar at both CO concentrations, the glucose concentration at 2400 ppm CO started at a higher level at 45 min, thus maintaining a higher level at 90 min. Plots of the change in glucose concentration 0--45 min versus the change in glu-
217
500 (+)
"t-'#-
400 '
d
[]
ETOHAlone
[] []
2400 ppm CO + ETOH 3000 ppm CO + ETOH
+++
Z 0 0 300 ._1 0 Z < -1I-- 200 LLI
0.05). With 3000 p p m CO, 5 of 10 rats died both with and without E T O H . At both CO concentrations, those rats that died, did so at a later time when they had received E T O H than when E T O H was absent. In this regard, a Chi-square analysis of the
100 90
(1)
[~
80
LU I-< rr" 7O _J < _> SO > rr 5O
3000 CO (10) 3000 CO+ ETOH (10) 2400 CO (7) 2400 CO + ETOH (10)
~ ~ _ _
"
"
,,~"
f/
40
10
I
I
I
I
I
I
10
30
50
70
90
110
0
f /
i
I
210
i
I"
330
TIME (min) Fig. 3. Survial rate of rats exposed to carbon monoxide for 90 rain. Starting number of animals in
each group indicated in parentheses.
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combined 2400 p p m and 3000 p p m mortality data at 45 min of CO exposure indicated a statistically significant difference (df = 1, P < 0.01). None of the 12 rats given E T O H alone died. The E T O H treated rats, at both CO concentrations, that died had significantly lower blood glucose at the time of death than those which survived after 90 min of CO exposure [(died, 2400 ppm, 9.1 mg/dl; 3000 ppm, 16.3 _ 6.1 mg/dl) (lived, 2400 ppm, 81.0 ± 13.6 mg/dl; 3000 ppm, 91.3 ± 24.0 mg/dl)]. Tests of mortality versus hypoglycemia using Chi-square at 2400 p p m and 3000 p p m CO gave 4.44 (df = 1, P < 0.05) and 6.67 (df = 1, P < 0.01), respectively. Hypoglycemia in this instance was defined as glucose concentration < 40 m g / d l [23]. Blood glucose in the rats destined to die was also considerably lower than survivors after 45 min of CO exposure. This association of severe hypoglycemia and death during CO poisoning was noted earlier (Penney et al., unpublished data). At both CO concentrations blood E T O H concentration tended to be higher in non-survivors at the time of death than after 90 min of CO exposure in survivors, but the differences were not significant. Moreover, those rats with the highest E T O H concentration tended to have the lowest blood glucose concentration (r = 0.51, P < 0.05). A m o n g the 3000 p p m rats treated with E T O H , non-survivors showed a much larger increase in hematocrit during the first 45 min of CO exposure (6.7 ± 0.1%0 vs. 3.6 ___ 0.6%).
Neurologic index Rats given E T O H alone displayed a neurologic index of 17.8 at the termination of E T O H infusion (90 min), reflecting their intoxicated condition (Fig. 4). Neurologic index then fell progressively until, by 24 h it was not differ-
[]
+++ (+) + ++
26 X HJ a Z
ETOH Alone
[]
2400 ppm CO Alone
[]
2400 ppm CO + ETOH
i
3000 ppm CO Alone
[]
3000 ppm CO + ETOH
o
16
w Z
12
10
Z
9
12 iii~4
i. 120
9O
PERIOD
24 hrs
240
OF CO EXPOSURE AND RECOVERY
(min)
Fig. 4. Neurologic index of rats following carbon monoxide exposure. Compared to the ethanol alone group: *, P < 0.05; *+, P < 0.01; ++*, P < 0.001. Compared to the 2400 ppm CO alone group: ( + ) , 1 S.E.M. Number of animals indicated at the base of columns.
220
ent from 6 (i.e. normal), suggesting that blood E T O H was near zero. Combined CO exposure and E T O H treatment increased neurologic index above that with E T O H alone, and with 2400 p p m CO after 90 min of exposure and after 120 and 240 min of recovery the increase nearly equalled the effect of E T O H alone at those times. With 3000 p p m CO, the increase in neurologic index at 120 and 240 min was somewhat greater than the effect of E T O H alone at those times. After 24 h of recovery, there was no significant difference in neurologic index between 3000 p p m CO rats receiving E T O H and those not receiving E T O H , suggesting that E T O H exerts neither a protective nor a deleterious effect on brain function at this CO concentration. Neurologic index was not determined at 24 h for rats exposed to 2400 p p m CO alone. Neurologic index was significantly higher at 240 min of recovery in rats exposed to 3000 versus 2400 p p m CO, indicating a doseresponse relationship between CO concentration and neurologic deficit. It should be noted that when deaths occurred, rats with the greatest potential neurologic deficit were eliminated, thus the neurologic index values from groups with high mortality are biased on the low side. Also, the regression plots do not contain data on rats which died during CO exposure, the ones which incurred the most severe hypoglycemia. Discussion Consistent with past reports of acute CO poisoning [19], we observed hypothermia, hypotension and evidence of hemoconcentration in the CO-exposed rats, which was not altered by E T O H treatment. The blood E T O H concentration attained in the rats was 2 - - 4 times the human legal intoxication level (100 m g / dl). However, it was well below the lethal blood level of 890--1000 m g / d l for rats receiving E T O H alone [24]. Whether the lethal E T O H level in the presence of CO is lower, is unknown. While not markedly altering the neurologic deficit obtained 24 h after CO poisoning, a time when neither CO nor E T O H were present in the animals, E T O H appeared to increase the survival rate at 2400 p p m CO and the tolerance time at both CO concentrations. If CO exposure had stopped before the 90 min m a r k (e.g. 45 min), the differences in survival rate between rats given E T O H and those not given E T O H would have been larger. Not only was blood E T O H concentration increased in the presence of CO, but the increase was related to CO concentration. E T O H exerted no effect on the average degree of hypoglycemia which developed late in CO poisoning, but the hypoglycemia was worsened by higher CO concentration. In contrast, the hyperglycemic rebound following CO exposure was increased by E T O H . Our results also show that early hyperglycemia was protective of later severe hypoglycemia, at both CO concentrations, whether or not E T O H was present. During recovery, the magnitude of the hyperglycemic rebound was directly related to the magnitude of the previous hypoglycemia, again at both CO concentrations, with or without E T O H . An examination of the data for the survivors and the non-survivors revealed severe hypoglycemia in the latter, and more or less normal blood glucose in the former, whether or not E T O H was present. The association of severe hypoglycemia and death has been noted earlier (Penney et al., unpublished
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data). Because the rats with the highest blood E T O H tended to be non-survivors, and those rats also tended to have lower blood glucose, E T O H would appear to be disadvantageous to survival in this regard, in some subjects. As is well known, CO exerts its toxic effects by avidly binding to hemoglobin. In so doing, it renders a fraction o f the hemoglobin incapable of transporting oxygen and decreases the ease of tissue oxygen unloading. Mainly due to the latter effect, an hypoxic state exists which is more severe than that of an hypoxic hypoxia of similar arterial oxygen saturation. Whereas previous studies described only a hyperglycemic response to CO poisoning [25,26], recent experiments suggest that the response is more complex. Hyperglycemia develops during early CO exposure, but is then attenuated or reversed during late CO exposure; a second hyperglycemic surge occurs during early recovery, followed by a return to normal values. We have termed this a "camel-back" pattern. Hypoglycemia is exacerbated by increased CO concentration, saline administration, and the prevention of CO-induced hypothermia (Sutariya et al., unpubl, data). Increased neurologic deficit has been correlated with a larger post-CO hyperglycemic rebound (Penney et al., unpublished data). Approximately 90--98% of ingested E T O H is oxidized by the liver, while the remainder is excreted unchanged [27]. As with other drugs [28], the rate of E T O H metabolism is dependent upon hepatic blood flow. Sylvester et al. [29] has shown that for a given level of arterial oxygen saturation, hypoxic hypoxia results in a relatively greater shunting of blood from the splanchnic vascular bed to the liver than does CO hypoxia. This could explain the higher blood E T O H concentration we observed in the CO-exposed rats. Topping et al. [30] report that 20% COHb saturation decreases E T O H utilization in the in vitro liver by one-half, and also decreases E T O H uptake. On the other hand, the hemoconcentration during CO exposure and the hemodilution following CO exposure, which we have inferred on the basis o f changes in hematocrit, should not have affected the plasma E T O H concentration, because E T O H is fairly uniformily distributed throughout all tissues and all fluids of the body [27]. CO-induced hemoconcentration was probably due to a redistribution of body fluids. Ogawa et al. [31] observed a 20% fall in blood volume after 30 min at 60% COHb saturation, mainly resulting from a sharp reduction in plasma volume caused by increased vascular permeability. Pohorecky and Newman [32] found that E T O H in doses of 1, 2 and 4 g/kg produced 4.7%, 7.3% and 15.3% increases in hematocrit, respectively, after 1 h. The highest E T O H dose decreased plasma volume by 20%, indicating that E T O H induces fluid loss from the vascular compartment. Nonetheless, we saw no evidence of hemoconcentration in our rats given E T O H alone, and no additional hemoconcentration occurred in the CO-exposed rats treated with E T O H . It is significant that E T O H treated rats which died during CO-exposure experienced greater increases in hematocrit than the survivors. This suggests that the vascular damage in the non-survivors was greater, but whether this was due to the hypoglycemia per se, is unknown. E T O H and other alcohols are reported to provide protection during hypoxic hypoxia [33,34]. This protective effect of E T O H could involve several different mechanisms: (1) an anesthetic depressant action lowering CNS metabolic rate
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[27], (2) an induction of hypothermia also reducing CNS metabolic rate, and (3) a specific metabolic action. A reduction in metabolic rate by whatever means would decrease the need for oxygen and prevent damage to sensitive aerobic organs, such as the brain. Sutariya et al. [6] showed that CO-induced hypothermia, or hypothermia produced by pre-cooling, improves survival and neurologic outcome in CO-poisoned rats. Lomax et al. [5] reported that ETOH-(0.5--3.2 g/ kg body wt) causes a dose-dependent fall in body temperature in rats. A 1.5 g/kg dose caused a fall of 1.6°C over 60 min at an environmental temperature of 18°C, not too different from the 1.1 °C decrease over 90 min observed in the present study at a 25°C room temperature. However, E T O H administration in our CO-exposed rats produced no greater hypothermia than in rats given CO alone, suggesting that CO's interference with body temperature control is much greater than that of E T O H , at least at the CO concentrations used in the present study. Some investigators [33] suggest that E T O H may provide benefit through development of ketosis, which by oxidizing ketones for energy production, minimizes the deleterious accumulation of brain lactic acid, since development of acidosis is postulated to play a major role in cell damage [35]. In this regard, significant increases in beta-hydroxybutyrate and glycerol have been reported in pigs infused with E T O H [36]. Low blood glucose and ketosis in fasted rats has been suggested to explain an increased tolerance of extreme hypoxia, as compared to fed rats [37]. E T O H is reported to decrease [17], increase [38], and not change [39] blood glucose in fed rats; however, we saw no hypoglycemia in our non-fasted rats given E T O H , and E T O H failed to exacerbate the hypoglycemic action of CO. Marks [40], in a review, suggests that E T O H may contribute to the development of hypoglycemia in two ways: (1) indirectly through its effects on hypothalamicpituitary-adrenal function, and (2) directly by impairing hepatic gluconeogenesis. Arky [41] suggests that gluconeogenesis is inhibited by E T O H due to the increase in the N A D H / N A D ratio, which inhibits the entrance of glycerol, lactate, and specific amino acids into metabolic pathways by which these metabolites are converted to glucose. CO hypoxia may do something similar. Hyperglycemic rebound with or without E T O H may occur for two reasons: (1) reversal of CO-hypoxia which occurs in rats within 2 h after transfer to room air [28], thus unblocking gluconeogenesis for the reasons described above, and (2) CO-stimulated release of catecholamines [42] acting on the liver to increase glycogenolysis and glucose release. In addition to these factors, insulin release is suppressed and glucagon and corticosteroid release is stimulated by E T O H [17,35,43]. Plasma insulin is also lowered by CO exposure (Penney et al., unpublished data). High blood E T O H concentration during recovery may have continued to perturb the levels of these hormones as indicated, providing an environment for development of greater hyperglycemia. Because catecholamine release is also stimulated by E T O H [44], E T O H could magnify the hyperglycemic response. However, we did not measure any of these hormones in our study. Other factors may also be involved. As observed in this experimental model the effects of CO are far more prominent than those of E T O H in terms of heart rate, blood pressure, hematocrit, and
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b l o o d g l u c o s e , at least at t h e d o s e levels o f C O a n d E T O H u s e d . E T O H a p p e a r s to p r o v i d e s i g n i f i c a n t s u r v i v a l p r o t e c t i o n f r o m C O p o i s o n i n g ; a s h o r t e r p e r i o d o f C O e x p o s u r e at t h e C O c o n c e n t r a t i o n s u s e d h e r e w o u l d h a v e p r o d u c e d n e a r l y 1 0 0 % s u r v i v a l in E T O H t r e a t e d a n i m a l s . S i n c e E T O H levels a r e e l e v a t e d in t h e presence of CO, might an individual ingesting ETOH, more rapidly reach the l e t h a l level in t h e p r e s e n c e o f C O ? O n t h e o t h e r h a n d , t h e p r e s e n c e o f E T O H d o e s n o t a l t e r t h e d e g r e e o r r a p i d i t y o f d e v e l o p m e n t o f h y p o g l y c e m i a , w h i c h is associated with death. However, ETOH does increase the hyperglycemic rebound, a l t h o u g h it d o e s n o t a p p e a r t o i n c r e a s e n e u r o l o g i c d e f i c i t d u r i n g r e c o v e r y .
Acknowledgements T h i s w o r k was s u p p o r t e d b y a r e s e a r c h g r a n t f r o m t h e A m e r i c a n H e a r t A s s o c i a t i o n o f M i c h i g a n . W e w i s h to t h a n k L i n d a M c C r a w f o r h e l p in p r e p a r a t i o n o f the manuscript.
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