Download Quantitative Estimation of Barbiturates in Blood by Ultra

QUANTITATIVE ESTIMATION OF BARBITURATES IN BLOOD BY
ULTRA-VIOLET SPECTROPHOTOMETRY.
II. EXPERIMENTAL AND CLINICAL RESULTS*
R. S. FISHER, M.D., J. T. WALKER, P H . D . , AND C. W. PLUMMER, B.S.
From the Department of Legal Medicine, Harvard Medical School, and the Chemical Laboratory of the Massachusetts Department of Public Safety, Boston, Massachusetts
The literature contains several reports of experimental studies relating concentrations of barbiturate in blood and tissue to the dosages administered.
There are, however, little data available with regard to concentrations obtained
after clinical use of these drugs. Also, clinical studies of the relation of blood
concentration of a barbiturate to the resulting degree of central nervous system
depression are not available. Such information is needed both to control clinical
administration of the drugs and to evaluate prognosis in cases of barbiturate
overdosage. The need has been especially emphasized by the advent of a relatively simple method of determining blood concentrations of barbiturates.
This paper contains the results of experimental and clinical studies on barbiturates using the analytical technic for the determination developed in these
laboratories.4
OBSERVATIONS
/. Experiment in Animals
Mongrel hounds of similar weight were given anesthetizing doses of sodium
pentobarbital and barbital by stomach tube. The blood concentrations of the
drugs were determined at intervals, and concomitant observations of the degree of
depression were made. The results are shown in Table 1. With pentobarbital
in a dose of 36.4 mg./Kg. (40 mg./Kg. as the sodium salt) the maximum blood
concentration of 2.2 mg./lOO ml. was reached in two hours, following which there
was a plateau maintained for several hours. By the sixth hour the blood barbiturate concentration was beginning to decrease. After twelve hours there was
partial return of consciousness with agitation and ataxia, and the blood concentration had fallen to less than 50 per cent of its maximum level. Only a trace
remained at twenty-four hours.
In contrast to these results are those when barbital, 200 mg./Kg., was given.
Here the blood barbiturate concentration continued to rise until the fourth hour,
after which it decreased gradually, but barbiturate was still present in considerable quantity twenty-four hours after administration. Of more significance is
the fact that with comparable effects on the central nervous system, the blood
concentrations of barbital were between 7 and 9 times as high as those of pentobarbital. This difference is indicative of the marked variation in physiologic
potency among the various barbiturates. It suggests, too, that the relative
"slowness of action" of barbital is in large part due to the purely mechanical or
* Received for publication, January 19, 1948.
462
4G3
BARBITURATES IN BLOOD
physical factors whereby a longer absorption time is necessary to attain the
blood concentrations that are required to produce a given effect with this drug.
The longer duration of effect would seem to be partly of the same "mechanical"
nature, although the ultimate mechanism of detoxication obviously is also of
major importance in this connection.
TABLE 1
BLOOD CONCENTRATION OF PENTOBARBITAL AND BARBITAL IN DOGS IN RELATION TO
DEPTH OP CENTRAL NERVOUS SYSTEM DEPRESSION
PENTOBARBITAL,* 36.4
HOURS AK1STRATION
Blood cone.
MG./KC
D e p t h of depression
mg./WO ml.
BARBITAL* 200 M C . / K G .
Blood cone.
D e p t h of depression
nig./ 100ml.
13. S
Still erect b u t with marked
ataxia.
2.2
Deeply comatose. Corneal
reHex absent. No response
to painful stimuli.
Same as 1. hr.
15.3
3
4
2.2
2.2
Same as 1 hr.
Same as 1 hr.
15. S
17.G
6
1.8
Same as 1 hr., beginning to
whine slightly.
16.2
.12
0.9
15.0
24
Trace
<.15
Eyes open. Thrashing about,
unable to stand, resists
venipuncture.
Up and about. Normal.
Comatose. Corneal reflex
active. No response to
painful stimuli.
Same as 2 hr.
Same as 2 hr. except corneal
reflex slightly loss active.
Corneal
reflex
active.
Whined and stirred a t
venipuncture.
Eyes open.
Thrashing
about but unable to stand.
1
l.S
2
11.S
Up and about but
marked ataxia.
with
* Given by stomach tube with 50 ml. of water per Kg. body weight after a twenty-four
hr. fast. Water intake was not restricted.
/ / . Clinical Experiments in Human Subjects
A series of patients, undergoing the "amytal test" for evaluation of blood
pressure response to sedation, was studied to determine the blood concentrations of amytal obtained. A second group of patients, who were receiving a new
barbiturate (5-ethyl-5-/3-methylall.ylbarbituric acid), under investigation as a
therapeutic agent in essential hypertension, was similarly studied. The results
are shown in Table 2.
Examination of the amytal results (Cases 1 to G) shows that even in this small
group of patients there was wide variation in the blood concentrations of amytal
obtained in different patients under clinical conditions which were as nearly
uniform as possible. This variation does not appear to be related to sex or
weight. Patients 1 and 2 were of the same sex, weight and age, and received
identical amounts of the barbiturate; they showed over 100 per cent difference
in blood amytal concentration (0.40 mg. per 100 ml. versus 0.91 mg. per 100 ml.).
464
FISHER, WALKER AND PLUMMER
The clinical observation that certain patients undergoing this test cannot be
readily aroused for some hours, whereas others remain conscious or sleep only
lightly, would seem to correspond with the observed variations in blood barbiturate levels. The second group of patients (7 to 11), who ingested 5-ethyl-5/3-methylallylbarbituric.acid, also showed a striking variation (from 0.26 to 1.00
mg. per cent) in blood concentrations after identical doses of the drug. Another
feature worthy of comment is the fact that the patients of the second group
TABLE 2
BLOOD CONCENTRATION
AFTER T H E R A P E U T I C D O S E S OF SODIUM AMYTAL AND
5-ETHYL-5-0-METHYLALLYLBARBITURIC
ACID
HOURS AFTER ADMINISTRATION
0
SEX
2
1
1
3
REFERENCE
NUMBER
WEIGHT
Dose
Dose
Accumulated
total
dose
Blood
cone.
Dose
Accumulated
total
dose
Blood
cone.
Sodium Amytal*
53
53
53
47
51
48
F
F
F
M
M
M
Kg.
mg./Kg.
mg./Kg.
mg./Kg.
mg./100
ml.
mg./Kg.
mg./Kg.
mg./100
ml.
63.1
61.9
70.6
79.5
71.S
85.S
3.2
3.2
' 2.S
2.5
2.7
2.3
3.2
3.2
2.S
2.5
2.7
2.3
6.4
6.4
5.6
5.0
5.4
4.G
0.25
0.66
0.38
0.33
0.2S
0.63
3.2
3.2
2.8
2.5
2.7
2.3
9.6
9.6
S.4
7.5
7.1
6.9
0.40
0.91
0.64
0.53
0.33
0.67
1—M.
2—R.
3-D.
4—S.
5—J.
6—J.
S.
S.
D.
L.
M.
E.
5-Ethyl-5-/3-Methylallylbarbituric Acidf
27
55
54
4S
31
F
M
M
M
M
51.2
76.4
72.2
76.4
73.2
1.9
1.3
1.3
1.3
1.3
1.9
1.3
1.3
1.3
1.3
3.8
2.6
2.6
2.6
2.6
—
0.70
0.70
0.51
0.29
1.9
1.3
1.3
1.3
1.3
5.7
3.9
3.9
3.9
3.9
0.43
0.75
1.00
0.73
0.26
7—E. A.
S—S. F.
9—S. B .
10—H. T .
11—C. G.
* Given 3 grain capsules of sodium amytal successively at " 0 " hi\, " 1 " hi'-, and " 2 " hi-.
Blood samples were drawn at time of third dose and one hour thereafter.
f Given i5-ethyl-5-/3-methylallylbarbituric acid on same schedule, but each dose consisted of 1.5 grains of the drug.
received only one-half as much barbiturate as those in the sodium amytal group,
but they exhibited higher blood barbiturate concentrations (average of three
hour amytal levels 0.50 mg. per cent; average 5-ethyl-5-|3-methylallybarbituric
acid 0.63 mg. per cent).
III.
Non-fatal Poisoning in Human Subjects
Several patients, who had ingested large doses of barbiturates with presumably
suicidal intent, were studied from the time of admission until they had recovered
consciousness. They are grouped in Table 3 according to the identity and ap-
TABLE 3
BLOOD
CONCENTRATION
AFTER
SUICIDAL
(OR
ACCIDENTAL) I N G E S T I O N OF N O N - F A T A L
D O S E S OF V A R I O U S B A R B I T U R A T E S
NAME
SEX
AGE
DRUG
AMOUNT
IIRS. AFTER
INGESTION
(Al'I'ROX.)
F
F
41
60
Seconal
Seconal
1.9
0.6
R. 0 .
F
2S
W. H .
F
34
1.0
Seconal
(plus several amytal
capsules and a few
phenobarbital t a b lets)
2.0
Pentobarbital
R . IT.'
M
21
Pentobarbital
O.S
Z. B .
F
23
Pentobarbital
?
W. R.
M
43
Phenobarbital
?
L. R.
D . I.
R. D .
F
F
M
00
26
40
3.2
Phenobarbital
?
Barbital
?
Barbital
(Quantity not -known.
Suicidal intent denied. I d e n t i t y of
drug recovered from
urine established as
barbital by mixed
melting point with
known sample of
barbital.)
CLINICAL STATE OF
INTOXICATION*
mg./lOO ml.
grams
y-
L. W.
N. P.
BLOOD
BARB.
CONCEN.
36
6
12
12i
IS
0.5
O.S
O.S
l.S
1.5
16
22
2S
3Si
4
10
2
16
4S
16
22
34
60
66
31
41
53
1.6
1.0
0.95
<0.5
1.4
O.S
1.4
1.6
59
'65
77
S3
S9
101
107
113
125
131
137
149
173
197
221
Awake
Awake
Awake
Comatose
Awake
12.S
11.S
11.2
10.4
5.S
20.6
19.1
16.4
Comatose
Comatose
Awake
Awake
Comatose
Awake
Comatose
Comatose
Awake
Comatose
Awake
Awake
Comatose
Awake
Deeply comatose
Deeply comatose
Deeply comatose
15.4
13.2
11.S
10.S
S.9
S.5
7.6
7.1
6.3
6.3
6.3
5.4
2.6
l.S
1.7
Deeply comatose
Deeply comatose
Comatose
Comatose
Comatose
Comatose
Awake
Awake
Awake
Awake
Awake
Awake
Awake
Awake
Recovered
* Graded a t throe levels, viz.: Deeply comatose, indicates total arcflcxia including loss
of respiratory and pupillary response on strong supraorbital pressure; Comatose, indicates
a state in which tendon and/or cutaneous reflexes were present but there was no response
to the spoken voice and no oral response to painful stimuli; Awake, indicates oral response
to spoken voice and includes several instances in which the patient was extremely drowsy
b u t could be awakened or was awake but not clearly oriented. No patients completely
free of barbiturate effects were included in this category.
465
466
FISHER, WALKER AND PLUMMER
proximate dosage *of the barbiturate. Blood concentrations and concomitant
observations of state of cerebral depression are indicated.
The first in the Seconal series (L. W.) had taken a dose sufficient to cause coma
of twenty-four hours' duration and had awakened prior to collection of the blood
specimen reported. Nevertheless, she was still confused and had slurring of
speech. The second patient (N. P.) was treated with stimulant drugs and, although quite drowsy at six hours, she did not become disoriented. The blood
level attained and the clinical state of the patient were similar in general to those
encountered in the patients undergoing the "amytal sedation tests" (Table 2).
In the third patient (R. 0.) the presence of an undetermined amount of phenobarbital in the mixture ingested probably led to a higher barbiturate level at
the time of awakening than would be obtained had the drug been Seconal alone.
In the pentobarbital cases the lowest observed blood barbiturate levels accompanied by coma were 1.0,1.4 and 1.4 mg. per 100 ml., and those subsequent to
or at the time of awakening were in the neighborhood of 1.0 mg. per 100 ml.
Inasmuch as no significant quantity of barbiturate was recovered in gastric
lavage of the second patient in this series (R. H.) and the first blood specimen
was obtained after four hours, it is probable that the concentration of 1.4 mg.
per 100 ml. is maximal for this dosage. Likewise our experience with this and
other short-acting barbiturates would lead us to believe that in patient W. H.
the maximal blood concentration had been passed prior to collection of the first
blood specimen at sixteen hours.
The phenobarbital and barbital patients may be considered together, and they
show quite a different magnitude of blood barbiturate concentrations than those
with the short-acting barbiturates. With the long-acting barbiturates the
return of consciousness at blood levels of 11.8 mg. per 100 ml. (W. R.) and 7.6
mg. per 100 ml. (R. D.) again illustrates their lesser physiologic or toxic potency
as compared with their more rapidly acting relatives. The second phenobarbital
patient, L. R., although comatose at the time of the blood barbiturate determination, was not deeply depressed and subsequently recovered. The fact that L. R.
was comatose with 10.4 mg. of phenobarbital per 100 ml. of blood, whereas W.
R. awakened with a blood concentration of 11.8 mg. of the same drug, may be
due to normal physiologic variation or to other factors. These include the age
and sex differences as well as the fact that L. R. had not used phenobarbital frequently prior to this hospitalization, whereas W. R. had been taking the drugin moderate dosage for a prolonged period.
In the case of R. D. it was possible to obtain much of the urine excreted during
the first six days of his hospitalization. These specimens were analyzed for
barbiturate, and the composite results appear in Figure 1. The graph of blood
barbital levels is a relatively smooth curve with the decrease in blood barbital
in the first two and one-half days of observation, averaging 0.2 mg. per 100 ml.
per hour, while in the second half of the observation period, the rate approached
0.05 mg. per 100 ml. per hour. The urine barbital concentration varied from
300 in the first specimen to 12.1 mg. per 100 ml. in the last. The total amount
of barbital recovered in the five day period amounted to 7.4 grams! In spite
467
BARBITURATES IN BLOOD
of the extremely high blood levels encountered and the large amount of barbital
ingested, the patient made an apparent clinical recovery and was discharged
from the hospital without gross evidence of residual cerebral damage.
BARBITAL
CONCENTRATION
IN BLOOD
10.0MO . / | 0 0 ML
0
300
BARBITAL
CONCENTRATION
IN URINE
200MO ./lOO
ML
100-
08.0TOTAL
6.0
BARBITAL
RECOVERED
IN URINE 4 L
30
40
50
~i
60
r
70
BO
90
100
110
120
130
140
ISO
HOURS AFTER INGESTION
FIG.
1. BLOOD AND U R I N E B A R B I T A L CONCENTRATIONS AND U R I N A R Y R E C O V E R Y OK
B A R B I T A L I N A C A S E OF B A R B I T A L P O I S O N I N G
The shaded areas in the lower curve represent increments of barbiturate in the
periods indicated.
IV. Human Subjects: Fatal Poisoning
In the routine work of these departments, deaths caused or contributed to by
barbiturates are frequently encountered. Table 4 presents a series of such
cases with the barbiturate concentrations determined in postmortem blood or
tissue. None of the subjects in this group showed evidence of significant disease
on complete postmortem examination.
The cases fall into 2 general groups based on blood barbiturate concentrations.
468
FISHER, WALKER AND PLUMMER
The first 4 subjects with barbiturate levels of 2.5 mg. or less per 100 ml. all showed
concomitant alcoholism, and neither the barbiturate nor the alcohol concentration would ordinarily be considered as fatal levels. In the third case (7820), the
lapse of time between ingestion of noxious agents and death was known accurately
to be only one and one-half hours. The rapid onset of coma with prompt death
in this case and the fatal outcome of the other cases, despite the absence of
ordinarily fatal concentrations of either alcohol or barbiturate, is impressive and
TABLE 4
BLOOD CONCENTRATIONS AFTER SUICIDAL OR ACCIDENTAL I N G E S T I O N
OF F A T A L D O S E S
OF
VARIOUS BARBITURATES
CASE
HISTORY
•
S759
8633
7820
9661
8905
9485
S691
9658
8926
7732
Adult. Found dead. Chronic alcoholic. Also taking pentobarbital. Blood alcohol 0.17 per cent.
Adult. Found dead. Alcoholic and habitual user of pentobarbital. Blood alcohol 0.37 per cent.
35 year old male. Ingested 0.8 Gm. of neonal and some whiskey.
Died one and one-half hr. later. Blood alcohol0.13 percent.
Adult female. Found dead in bed with bottle of pentobarbital capsules at bedside. Blood alcohol 0.22 per cent.
Adult male. Admitted to hospital in coma. Died without
regaining consciousness forty-eight hr. later.
Female 29 years of age. Found dead in woods. Missing eight
days. All organs showed marked decomposition.
Adult female. Barbiturate suicide.
Barbiturate suicide. Found dead in hotel bathroom.
Stomach contained several grams of barbiturate.
Barbiturate suicide. Died two days after ingestion of unknown quantity of phenobarbital and pentobarbital. Analysis of brain tissue.
Ingested large amount of barbiturate in bathroom. Walked to
living room, collapsed and died in thirty minutes. Barbiturate identified as pentobarbital.
BARBITURATE
LEVEL
DETERMINED
mi./100
ml.
0.6
0.7
1.0-2.0*
2.5
4.8
6.6
7.2
12.0
12.3
58.0*
* Indicates barbiturate determinations by Stas Otto-gravimetric procedure prior to
development of present technic.
should focus attention on the very real danger of prescribing heavy barbiturate
sedation for alcoholics who are trying to "taper off". This phenomenon has
been commented on elsewhere in connection with experimental studies, 3 and
three, deaths thought to be due to the "combined effects" of barbiturate and
alcohol were reported from these laboratories in 1943 ? We believe that a search
for barbiturates, using the new technics that detect small amounts of the drugs
in "alcoholic" deaths, will reveal that the "combined effects of barbiturate and
alcohol" are frequently the true cause of death. The implications in the clinical
treatment of alcoholism are obvious.
BARBITURATES IN BLOOD
469
The remaining 6 cases are in general similar to those of Gettler 1 and illustrate
fatal blood concentrations of barbiturates. With the exception of Case #9485
all are known to have succumbed within three days of ingestion of the drug.
Undoubtedly, in some individuals who survive longer, lower postmortem barbiturate levels will be encountered. We have observed fatality where Seconal
poisoning was the only clinically evident cause of death, with the blood Seconal
concentration being less than 1.0 mg. per 100 ml. on the fourth day of coma.
Death ensued on the sixth day, but inasmuch as a postmortem examination was
not obtained, the case is not included in Table 4.
SUMMARY
1. The concentration of barbiturate in the blood (determined by a new ultraviolet spectrophotometric technic) has been investigated with regard to the
kind and dose of the drug and to the lapse of time after ingestion in a series of
non-fatal and fatal cases (therapeutic, accidental, suicidal).
2. Coma may occur with blood concentrations as low as 1.0 mg. per 100 ml.
after ingestion of certain short-acting barbiturates, whereas consciousness may
be regained after ingestion of a slow-acting barbiturate with a residual blood
concentration as high as 11 mg. per 100 ml.
3. Recovery from barbiturate poisoning (barbital) was observed in one instance in which more than 7.4 Gm. of the drug was excreted in the urine during
the course of six days.
4. Examples are presented in which death was apparently due to the combined
effects of alcohol and barbiturate, each of which was present in concentrations
not ordinarily considered fatal. The danger incident to indiscriminate use of
the two agents at the same time is stressed.
Acknowledgments.
We wish t o acknowledge our indebtedness to t h e various members of
the medical and surgical staffs of t h e Massachusetts Memorial, Boston City, Boston Psychopathic, New England Deaconess and Beth Israel Hospitals and of the Medical Examiner's Office, First District, Middlesex County, Massachusetts, who obtained specimens
the analyses of which are included in this report.
REFERENCES
1. GONZALES, T . A., VANCE, M., AND H E L P E R N , M . : Legal Medicine and Toxicology, New
Ed., New York: D . Appleton-Century Company, 1940, 754 p p .
2. J E T T E R , W. W., AND M C L E A N , R . : Poisoning by t h e synergistic effect of phenobarbital
and ethyl alcohol. Experimental study. Arch. P a t h . , 36: 112-122, 1943.
3. RAMSEY, H . , AND H A A G , H . B . : T h e synergism between the barbiturates and ethyl
alcohol. J. Pharmacol, and Exper. Therap., 88: 313-322, 1946.
4. W A L K E R , J. T., F I S H E R , R . S., AND M C H U G H , J. J . : Q u a n t i t a t i v e estimation of barbit-
urates in blood by ultra-violet spectrophotometry.
Clin. P a t h . , 1 8 : 451-461, 194S.
I. Analytical method.
Am. J.