Published May 20, 1943
T H E E F F E C T OF LOW O X Y G E N T E N S I O N ON TISSUE M E T A B O L I S M
(RETINA)
BY FRANCIS N. CRAIG AXeDHENRY K. BEECHER*
(From the Anesthesia Laboratory of the Harvard Medical School at the Massachusetts
General Hospital, Boston)
(Received for publication, February 27, 1943)
I
II
The retinas, obtained from three or six mongrel white rats, were prepared in the
light at room temperature, cut in half, and pooled. 5 minutes per rat were required
for preparation. The retinas were suspended in a medium that contained NaC1
0.118 ~, KC1 0.0024 ~, CaC1, 0.0017 ~r, MgC1, 0.0007 M, and glucose 0.011 ~ in
addition to the buffer. In medium containing phosphate buffer (Nail,P04 0.003 ~a
and Na2HPO, 0.017 ~), oxygen uptake was determined by the first method of Warburg
(1923). The lactic acid that accumulated in the vessel during the preliminary period
of equilibration and the experimental period, was determined colorimetricaUy by the
* Aided by a grant from the Milton Fund of Harvard University.
467
The Journal of General Physiology
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These experiments were undertaken initially to determine whether confirma"
tion could be found for the observation of Laser (1937 a) that in an atmosphere
containing only 5 volumes per cent of oxygen, both respiration and glycolysis
of rat retina were at or near their maximum rates. On the basis of his experiments with retina and other tissues Laser stated t h a t " the Pasteur effect under
the conditions of the experiments depends upon the oxygen tension rather than
upon the rate of respiration." Thus the implications of his observation are
such that it is important that they be either supported or challenged. Stern
and Melnick (1941) repeated the experiment of Laser while determining the
physiological absorption spectrum of the Pasteur enzyme, but the data were
obtained at a temperature of 27 °. For this reason it was desirable to have further data at 38 ° .
Since manometric methods alone were employed by Laser, his work provided
data for glycolysis in bicarbonate medium but not in phosphate medium. The
nature of the buffer has been found to influence profoundly the metabolism of
retina, particularly with respect to its magnitude and to its sensitivity to cyanide and carbon monoxide (Laser, 1936, 1937a, and 1937 b). Therefore it
was also of interest to test chemically whether the substitution of phosphate
for bicarbonate buffer would affect the relationship between glycolysis and
oxygen tension.
Published May 20, 1943
468
EFFECT OF LOW OXYGEN TENSION ON TISSUE METABOLISM
III
The data are summarized and compared with those of Laser in Table I. I n
phosphate medium the only significant change in Qo2 with time was an increase
from the 1st to the 2nd hour with 0.5 per cent oxygen; in bicarbonate medium,
the metabolism often fell off during the 2nd hour.
The data for bicarbonate medium are in essential agreement with those of
Laser. Therefore not so m a n y bicarbonate buffer experiments were done as
with phosphate, where we differ with him. I n bicarbonate medium there was
no significant decrease in oxygen uptake with oxygen mixtures between 95 per
cent and 5 per cent, while at 5 per cent the lactic acid output was almost at the
anaerobic level.
I n phosphate medium, oxygen uptake was more sensitive to oxygen tension.
Laser reported no change in oxygen uptake when the oxygen tension was
reduced from 100 per cent to 5 per cent, whereas in our experience, oxygen
uptake was 38 per cent less at 10 per cent oxygen than at 100 per cent, and 51
per cent less at 5 per cent oxygen. The Qo, in 100 per cent oxygen, however,
agreed with his figure of 17. When the individual data for respiration at
different oxygen tensions were plotted against the corresponding data for
glycolysis in Fig. 1, a linear relationship was suggested. This is in marked
contrast to the situation in bicarbonate medium, where respiration and glycolysis varied with oxygen tension independently of each other.
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method of Barker and Summerson (1941). In nine tests the lactic acid formed in
the equilibration period at different oxygen tensions was 25 4- 1.8 per cent of the total
found at the end of a 2 hour experiment. Accordingly, 75 per cent of this total was
used in calculating the Qa. Qa -- 0.004 mg. of lactic acid, per milligram of tissue per
hour. In medium containing bicarbonate buffer (NaHCO~ 0.024 M and 5 per cent
CO~), both oxygen uptake and lactic acid output were determined by the second
method of Warburg (1924). In this method the estimation of lactic acid output
includes the assumption that the respiratory quotient is 1. Actually the mQ. of
retina is lower (Laser, 1937a; Greig, Munro, and Elliott, 1939), but this fact does not
impair the usefulness of the method.
The gas mixtures were made by displacement of saturated CaC1, solution from 20
liter carboys. They were analyzed with the Haldane apparatus. The mixtures were
reported on a dry basis, it being understood that the water vapor at 38°, the temperature of the experiments, amounts to 7 volumes per cent. Consequently the tension
in atmospheres is equal to 0.93 times the volume per cent as stated in the body of the
paper. Laser's procedure was followed particularly with regard to the rate of shaking
(160 cycles per minute) and, in bicarbonate medium, to preliminary equilibration of
the vessels with the gas mixtures before adding the tissues. At the conclusion of a 1
or 2 hour experimental period, the retinas were rinsed and dried to constant weight.
When lactic acid was to be determined chemically, the experiment was conducted for
2 hours to obtain a larger amount for analysis.
Published May 20, 1943
FRANCIS N. CRAIG AND HENRY K. BEECIt'ER
469
IV
B u r k (1939) has discussed a n d r e s t a t e d three m a i n theories to explain the
observed fact t h a t in m a n y kinds of living ceils less alcohol or lactic acid accumulates in the presence of air t h a n when oxygen is excluded. I n brief t h e y
TABLE I
Oxygen Uptake and Lactic Acid Output of Retina
In bicarbonate medium the data are for the 1st hour only. In phosphate medium the
data represent the average for 2 hours, in our experiments.
Oxygentension
Vol.per cent
Laser's
(1936,1937a)
retina data for
QO*
1
QG
The present
data with standard errors of the means.
No'°f
I
observations
QO,
I
QG
Bicarbonate medium
30
32
45.0
10-12
29.2
35.0
68.1
70.8
3-6
27.85
29.0
71.0
67.1
0-0.5
5
88
34 4. 4.6
41 4- 0.9
27 -4- 3.9
72 4- 2.9
34-0.4
77 4- 4.3
Phosphate medium
100
17.5
10
17.4 4- 1.0
204- 1.7
10-12
17.5
7
10.8 4- 0.7
26 4- 2.4
3-6
19.75
17.90
7
8.5 4- 1,1
37 4- 1.6
10
1.3 4- 0.4
44-4-2.3
0.5
are first, "the f e r m e n t a t i o n intermediates are oxidized (Pasteur, Pfeffer,
Pfluger)," second, "the f e r m e n t a t i o n intermediates are m a i n l y resynthesized
(Meyerhof)," a n d third, "the f e r m e n t a t i o n intermediates are m a i n l y n o t
f o r m e d " because " o x y g e n p r e s u m a b l y m a i n t a i n s some reduced c o m p o n e n t of
the f e r m e n t a t i o n enzyme system in a n oxidized inactive f o r m " (Lipmann,
1942). T h e results of Laser in b i c a r b o n a t e m e d i u m , which our experiments
confirm, s u p p o r t the third theory, for t h e y show t h a t the r a t e of glycolysis
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95
Published May 20, 1943
470
E]?FECT OF LOW OXYGEN TENSION ON TISSUE M]~TABOLIS~vr
depended in these circumstances on the oxygen tension and not on the rate of
respiration.
A marked increase in the sensitivity of oxygen consumption to low oxygen
tension was observed when phosphate medium was substituted for bicarbonate.
The extent to which this change can be accounted for on the basis of a specific
influence of the buffer on diffusion, is not known.
In connection with his studies on retina and other tissues, Laser (1937a)
called attention to the fact that a 5 per cent oxygen mixture is nearer to the
physiological oxygen tension of the tissues than is 100 per cent oxygen, corn-
1.0
~O O C I ) ~
0
.8
6
o
4
>,
©
2
0
I
0
I
f
I
4
.6
RESPIRATION
2
I
I
.8
1.0
FIG. 1. Oxygen uptake at different oxygen tensions as a fraction of the rate in 100
per cent oxygen in each experiment plotted against the corresponding value for lactic
acid output as a fraction of the rate in 0.5 per cent oxygen.
monly employed in experiments on surviving tissue. He believed also, with
Warburg, that tissue metabolism was largely independent of oxygen tension.
Since our data on oxygen consumption in phosphate medium, in contrast to
Laser's results, show marked sensitivity to oxygen tension, it m a y be well to
mention other instances of sensitivity to oxygen tension. The ingenious experiment of Laser (1932) with inflated rat lungs supported in the gas phase of
the Warburg flask, should be recalled. Here, in a situation in which questions
of diffusion and buffer specificity do not arise, the Qo~ decreased from 7.1 to
4.6 when the oxygen tension changed from 100 per cent to 10 per cent. More
recently, Kempner (1939) has criticized the Warburg point of view, and since
then oxygen uptake-oxygen tension curves with critical oxygen tensions well
above 5 per cent have been published for bone marrow (Warren, 1942), kidney
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0
o
Published May 20, 1943
]~RANCIS N. CRAIG AND H E N R Y K . B E E C t t E R
471
(Laser, 1942), and brain cortex, medulla, and spinal cord (Craig and Beecher,
1943). Of these, bone marrow and brain cortex exhibit also the linear relation
between respiration and glycolysis just described for retina in phosphate buffer.
One implication of this relation is that oxygen tension may act on the two
processes through the mediation of a common agent.
SUMMARY
We are indebted to Dr. Fritz Lipmann for many helpful suggestions during
the course of this study and to Miss Anna Murphy for her careful assistance
with the chemical determinations.
CITATIONS
Barker, S. B., and Summerson, W. H., The colorimetric determination of lactic acid
in biological material, J. Biol. Chem., 1941, 138, 535.
Burk, D., A colloquial consideration of the Pasteur and neo-Pasteur effects, Cold
Spring Harbor symposia on quantitative biology, Cold Spring Harbor, Long
Island Biological Association, 1939, 7, 420.
Craig, F. N., and Beecher, H. K., The effect of oxygen tension on the metabolism of
cerebral cortex, medulla, and spinal cord, J. Neurophysiol., 1943, 6~ 135.
Greig, M. E., Munro, M. P., and EUiott, K. A. C., Metabolism of lactic and pyruvic
acids in normal and tumour tissues. VI. Ox retina and chick embryo, Biochem.
J., London, 1939, 33,443.
Kempner, W., The r61e of oxygen tension in biological oxidations, Cold Spring Harbor
symposia on quantitative biology, Cold Spring Harbor, Long Island Biological
Association, 1939, 7, 269.
Laser, H., Manometrische Atmungsmessungen an der intakten Warmbluterlunge,
Biochem. Z., Berlin, 1932, 248, 9.
Laser, H., The metabolism of retina, Nature, 1936, 136, 184.
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Lactic acid production by rat retina in a medium containing phosphate was
studied chemically. One half as much lactic acid was found as in a medium
containing bicarbonate. In our experience the rate of respiration in a phosphate medium was sensitive to oxygen tension, for it was 38 per cent lower at
10 per cent and 51 per cent lower at 5 per cent oxygen than at 100 per cent
oxygen. Previously Laser had reported no decrease in respiration at 5 per cent
oxygen in phosphate medium. In phosphate medium, when the oxygen tension
was varied, respiration and glycolysis bore a reciprocal relationship to each
other.
In bicarbonate medium, when the oxygen tension was lowered from 95 per
cent to 5 per cent there was no significant change in the respiration, but glycolysis was increased nearly to the anaerobic level. This agrees with the earlier
experiment of Laser in bicarbonate medium and adds support to his conclusion
that the rate of glycolysis is controlled by oxygen tension rather than by the
rate of respiration, under the conditions of the experiment.
Published May 20, 1943
472
EFFECTOF LOW OXYGEN TENSION ON TISSUE ~IETABOLISM
Laser, H., Tissue metabolism under the influence of low oxygen tension, Biochem. J.,
London, 1937a, 31, 1671.
Laser, H., Tissue metabolism under the influence of carbon monoxide, Biochem. J.,
London, 1937b, 31, 1677.
Laser, H., A critical analysis of the tissue slice method in manometric experiments,
Biochem. J., London, 1942, 36~ 319.
Lipmann, F., Pasteur effect, in A symposium on respiratory enzymes, Madison, The
University of Wisconsin Press, 1942, 48.
Stem, K. G., and Melnick, J. L., The photochemical spectrum of the Pasteur enzyme
in retina, J. Biol. Chem., 1941, 139, 301.
Warburg, 0., Versuche an fiberlebenden Carcinomgewebe, Biochem. Z., Berlin, 1923,
142, 317.
Warburg, O., Verbesserte Methode zur Messung der Atmung und Glykolyse, Biochem.
Z., Berlin, 1924, 152, 51.
Warren, C. O., The Pasteur effect in bone marrow, with particular reference to the
results obtained by different methods, J. Cell. and Comp. Physiol., 1942, 19, 193.
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