ETHER ANESTHESIA. That the alkali reserve during ether anesthesia is reduced from 13 to 34 ...

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ETHER ANESTHESIA.
III. R6LE OF LACTIC ACID IN THE ACIDOSIS OF ETHER
ANESTHESIA.
BY ETHEL RONZONI, IRENE KOECHIG, AXD EMILY P. EATON.
(From the Laboratory of Biological Chemtisry, Washington University School
of Medicine, St. Louis.)
(Received for publication, July 2, 1924.)
That the alkali reserve during ether anesthesia is reduced from
13 to 34 volumes per cent and that this condition is accompanied
by an increase in the hydrogen ion concentration of the blood have
been shown by a number of investigators, Van Slyke, Austin, and
Cullen (1922). It has recently been shown that these changes
take place early in anesthesia and that anoxemia alone is not
responsible (Cullen, Austin, Kornblum, and Robinson (1923)).
That they are not due to the production of acetone bodies has been
demonstrated by Leake, Leake, and Koehler (1923), and by
Gram& (1922) who found no acetone during prolonged anesthesias.
Leake interprets his findings on acetone bodies as evidence against
the formation of other acid products. This would leave with-
drawal of base from the blood as the alternative explanation.
The accumulation of lactic acid in the blood does not seem to be
excluded as the possible cause of changes in the acid-base
equilibrium. The importance of lactic acid in the acidosis of
muscular exercise and the influence oxidative processes have on
its accumulation suggest it as possibly responsible for these
changes.
In this paper an attempt has been made to correlate changes
in the lactic acid content of the blood with changes in the acid-
base equilibrium, and at the same time to show whether or not
the amount in the blood depends on the pH as controlled by the
CO2 tension, in this condition, as has been found by Anrep and
Cannan (1923) in experimental acidemia and alkalemia. An
effort was made to throw light on the possible source of lactic
465
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Ether Anesthesia. III
acid and the cause of its accumulation. The degree of cyanosis
or the oxygen content of capillary blood is the nearest index
we have of the oxygen supply to the tissues. A comparison of
oxygen content of arterial and venous blood has been studied in an
attempt to determine the condition of tissues in regard to the
oxygen supply.
Procedure.
Animals.-Dogs, weighing between 10 and 15 kilos, were used, a size
that could be easily handled by two persons and sufficiently large that the
withdrawal of 150 to 206 cc. of blood would not seriously disturb the con-
dition of the animal. The animals were fed as usual on the the day preced-
ing, but received no food on the day of the experiment. They were placed
in a cage the evening before and kept as quiet as possible until the experi-
ment was started. The dogs were tied to the board, usually without strug-
gling, and the initial sample of blood was taken. Ether was then adminis-
tered. In all cases the normal temperature of the dog was maintained by
means of a heating pad controlled by a thermostat placed in the rectum
of the dog, as described by Bishop (1923).
Induction of Anesthesia.-Anesthesia was induced by the drop method.
A wire cone covered with gauze was placed over the nose of the animal.
Even with the greatest care we found ourselves unable to produce anesthesia
without some excitement and struggling on the part of the animals, Experi-
ments I, II, and III. To reduce this to a minimum we found the beet
method was to decrease the period of induction. Since the concentration of
ether in the blood depends on the alveolar tension, and since the stimulus
to respiration occasioned by increasing the CO1 tension had already been
shown to increase the rate of elimination of ether (Haggard and Henderson
(1919), White (1923), Ronzoni (1923)), it naturally occurred to us that in-
duction could be hastened in thesame way. This has recently been demon-
strated by Haggard (1924) to be true. The CO, concentration was increased
by using a close fitting mask and allowing the animal to rebreathe into
the mask until the rate of respiration was increased before pouring on the
ether. If the respiration was first stimulated there was no reflex inhibition
due to irritation of the mucous membrane of the nasal passage by ether.
This procedure also decreased the oxygen tension, but since the whole
procedure was over in a period of about 2 minutes this was thought to have
little effect on the subsequent blood reactions. Analysis of the air in the
mask showed from 6 to 8 ~01s. per cent CO1 and from 15 to 18 ~01s. per cent
Oz. The struggling was vigorous, but of short duration.
After induction, tracheotomy was performed and the animals were at-
tached to the ether apparatus described in a previous paper (Ronzoni
(1923)). If the animal was to recover the mask was replaced byafew layers
of gauze t.o insure adequate ventilation and the drop method continued.
In Experiments VI and VII the mask was used. The oxygen unsaturation
E. Ronzoni, I. Koechig, and E. P. Eaton 467
of the blood in the early part of these experiments shows that the ventila-
tion was interfered with. In Experiment VIII no mask was used. The
increased Oz unsaturation was due to depression of respiration accomr
parrying the deep anesthesia.
Collection of Samples.-Blood was drawn from the femoral artery into a
syringe previously coated with and containing about 4 inch of oil. The
initial sample in each case was taken through the skin and in those cases
where the animal was allowed to recover subsequent samples were drawn
in the same manner. Otherwise the artery was exposed and the blood taken
by needle and syringe. When removing the needle the circulation was
stopped for a few seconds by gentle pressure, thus allowing time for the
closing of the puncture hole. The blood was then allowed to resume its flow.
This procedure necessitated no part of the body being cut off from the
general circulation for more than a few moments.
The blood was transferred from the syringe under oil to a tube contain-
ing enough neutral oxalate to make 0.3 per cent and sodium flouride to give
a concentration of 0.05 per cent, shown by Evans (1922) to prevent produc-
tion of lactic acid. After collection the blood was placed in the refrigerator
until analyzed. That’ there was no significant change in the blood during
the interval between drawing and analysis is shown by the fact that there
was no measurable change in pH. Blood to be usedforetherdeterminations
was drawn directly into a pipette graduated in cubic centimeters. After
enough blood for duplicate determinations was collected this was measured
directly into aeration flasks containing oxalate. The last portion of blood,
the layer exposed to the air, was discarded. The aeration tubes of the
flasks were closed until aeration was started-within a few minutes after
drawing the blood.
Blood gas analyses were made on whole blood in the Van Slyke constant
pressure apparatus, using the technique described by Van Slyke and Stadie
(1921). The 02 and CO, contents of the blood as drawn were determined.
A 5 cc. sample was equilibrated with an air and CO2 mixture, containing
approximately 5 ~01s. of COZ and 19.5 vols. per cent 02. Theseequilibrations
were carried out in liter bottles at a temperature between 36” and 38°C.
The blood was removed under oil without exposure to air. The gas mixture
was trapped in the bottle and drawn out into a Haldane gas apparatus
under reduced pressure for analysis. Thus the actual tension of CO, and
02 with which the blood was in final equilibrium was determined. Analysis
of a sample of blood so equilibrated gives both the CO, and O2 capacity of
the blood, proper correction being applied for the amount of physically
dissolved 02. All gas analyses were made in duplicate, usually by two
observers.
The possible effect of ether contained in the blood on the blood gas
determinations suggested itself to us early in the investigation. We found
that concentrations of ether similar to those existing in the blood of an-
esthetized animals caused a considerable error in the CO2 determinations
as has also been reported by Austin (1924). An investigation of the condi-
tions of our elrperiments shows this error to be actually negligible. As
468 Ether Anesthesia. III
already described the blood was collected in an oil-coated syringe and
allowed to stand for some time under oil before analysis. The tubes used
for collection were 2 cm. in diameter, and, since whole blood was used,
stirring was necessary to mix the corpuscles and plasma before each sample
was measured. Examination of the blood after the samples had been
removed for gas analyses showed a great reduction in the amount of ether
in the blood. This is to be expected from the fact that the partition co-
efficient for ether between oil and blood is greatly in favor of the oil. Table
I gives data from a series of bloods examined; the maximum ether found in
blood at the time the gas analyses were made was 45 mg. per 106 cc. This
amount of ether added to a bicarbonate solution of a known strength in-
creased the apparent volume of COZ, obtained from 1 cc. of the solution,
by about 0.01 cc. If the total volume.of CO1 contained in 1 cc. of blood
amounted to 0.5 cc., then the error due to COz would be 2 per cent and the
apparent CO, content would be 2 per cent too high. The lower the CO*
content the greater the error due to ether. Since the CO2 content of blood
TABLE I.
Amount of Ether Contained in 100 Cc. of Blood, Calculatedfrom the Distri-
bution Ration and Relative Volumes of Blood and Air.
Ether per 100 cc. of blood.
When drawn. After equilibration.
ml. w7. WT.
162 45.0 11.3
183 41.3 12.7
196 38.5 13.7
132 40.2 9.4
as drawn falls as low as 25 vols. per cent the error might be increased to
4 per cent.
In the equilibration for the determination of CO* capacity the volume
of gas with which the blood was in equilibrium was 266 times that of the
blood used. The distribution ratio for ether between air and blood at 38”
is 1: 15. So the blood after equilibration would contain only l/14 the total
ether, or in the case of blood originally containing 162 mg. per 100 cc.,
11.3 mg., an amount too small to have a measurable effect.
Values for CO2 content of blood as drawn may therefore be from 2 to 4
per cent too high; and the apparent changes less than those really existing.
These errors would be reflected in the calculated values of CO, tension.
Such errors fall easily within the limits of error in lactic acid determina-
tions. The values for CO, capacity of the blood are not influenced by ether
since the amounts present caused no measurable effect in CO, determination.
The pH of the blood was determined by Cullen’s (1922) calorimetric
method. Clausen’s (1922) method was used for lactic acid. This deter-
E. Ronzoni, I. Koechig, and E. P. Eaton 469
mination often had to be left till the following day. In all cases the blood
filtrates, however, were made immediately after drawing the blood, the
sugar was precipitated, the filtrate placed in the refrigerator, and analyses
were made within the next 24 hours, a procedure which has been found tobe
safe. To eliminate the possibility of the ether in the blood affecting the
determination of lactic acid or the possible influence of acetone bodies,
the filtrates after having been measured for lactic acid determinations
were acidified and aerat.ed for 15 minutes at a temperature of 100”. Sam-
ples that were aerated showed the same lactic acid content as similar samples
unaerated, which shows that the ether has no effect and that acetone does
not accumulate. In fact, the lactic acid of the aerated sample always ran
a trifle higher than that of the unaerated. The differences were well within
the limits of error of the lactic acid method, but were always in the same
direction, an observation which we are unable to explain.
Ether determinations were made by a modification of the Nicloux method
described by Shaffer and Ronzoni (1923). Blood sugar determinations
were made on all samples, but these results will be discussed in a future
paper.
Calculation. of Data.
In all casest he pH and COz content of the blood were determined.
The COz tension was calculated by means of Hasselbalch’s equation
BHCOa
pH = pI<r + log H
2 8
crp 100
H&O8 = 760 = 0.01316crp
BHCOs = COz - 0.1316 crp
CO* iiustin, Cullen, Hastings, Mc-
COa tension = vgy2) Peters, and Van Slyke
0.1316 cx (lOnH-n”L + 1)
>
p is partial pressure of COz. For at the solubility coefficient of
COz in whole blood, Bohr’s (1905) value 0.511 was used. 6.20 was
taken as the value of pK1, this having been determined on dog’s
blood by Van Slyke and coworkers. Samples of the same bloods
were also equilibrated with a known tension of COz and the CO2
capacity was determined. This gives the data for the general
slope of the absorption curve of fully oxygenated blood when
correction is made for O2 unsaturation of the ‘blood as drawn.
These were plotted on CO2 diagrams introduced by Haggard and
Henderson (1919) and the volume per cent COz read off at a
470 Ether Anesthesia. III
constant CO* tension 38 mm. for purposes of comparing the changes
occurring in the alkali reserve. Corrections for the oxygen
unsaturation of the arterial blood as drawn were made by the
formula of Peters, Barr, and Rule (1921), K X Hb = D, where
K is a constant, Hb is oxygen unsaturation expressed in volumes
per cent, and D is the change in level of absorption curve expressed
in volumes per cent of COz. The value for K of 0.27 was used as
9
r.10
/
7.00
/
PC4 10 LO 30 40 SO 60 i 10 Mm. t
l CO2 content as drawn.
o Corrected for Oz unsaturation.
X Equilibrated sample.
CHART I. Data from Experiment VII.
determined by Doisy, Briggs, Eaton, and Chambers (1922).
Chart I, from the data of Experiment VII, shows the graphic
method.
The changes in pH, CO2 tension, alkali reserve at a constant
CO2 tension, and lactic acid, expressed in terms of lowering of
COz capacity, are plotted for all experiments. Protocols are given
in the tables and show the determined and calculated values on
which the diagrams are based.
E. Ronzoni, I. Koechig, and E. P. Eaton 471
RESULTS AND DISCUSSION.
Relation of LacticAcid to Changes inAlkali Reserve.-The accumu~
lation of lactic acid in the blood during anesthesia accompanier;
the fall in pH and in the alkali reserve as measured by the CO*
capacity at a constant CO2 tension. The relation between lactic
acid increase and the fall in CO, capacity of the blood may be seen
in Charts II, III, and IV, and is summarized in Table II. Assuming
that 4 mg. of lactic acid reduce the COz capacity 1 volume per cent,
the accumulation of lactic acid does not correspond to the fall
in alkali reserve. That this is an important factor, however, is
obvious. It is not to be expected that even though the accumula-
TABLE II.
Fall in CO2 Capacity Determined and Calculated from Lactic Acid.
-
T- I
Fall in CO% capacity.
I dctic acid - 1D fXV%l8C , Durntion
increase. Dmeinteerd- . lelculnted. Deiffnecre- . in pH. of anesthesia.
_
ma. c 01. PCI Cwal 01. per cen1 per cent hr.% min.
II 50.6 18.7 12.6 -33.2 0.49 5 0
III 33.7 15.3 8.4 -44.1 0.29 1 45
IV 49.2 12.2 12.3 +1.0 0.25 3 40
v 37.0 9.0 9.2 +2.2 0.24 2 5
VI 41.9 15.4 10.5 -31.8 0.25 2 5
VII 64.0 13.0 16.0 +23.1 0.27 2 0
VIII 30.5 12.0 7.6 -36.7 0.37 1 0
IX 41.9 10.5 10.5 zko.0 0.18 2 0
tion of lactic acid was entirely responsible for the change in CO2
capacity and pH, the amount in the blood at any one time would
exactly correspond, since undoubtedly the lactic acid is produced in
the tissues and must be in higher concentration there than in
the blood until equilibrium had been reached. At any time there
might be a withdrawal of base from the blood to the tissues without
a corresponding increase of lactic acid apparent in the blood.
Recent experiments of Stehle and Bourne (1924) show that
phosphoric acid leaves the muscles during anesthesia and sojourns
in the liver until the reassumption of kidney function after the
recovery of the animal; and since the transport of this must be
through the blood, it is suggested that the increased acidity of the
blood is due to the discharge of phosphoric acid from the muscle.
472 Ether Anesthesia. III
E. Ronzoni, I. Koechig, and E. P. Eaton 473
I 1. -.
I
474 Ether Anesthesia. III