J. comp. Physiol. 85, 15--24 (1973)
9 by Springer-Verlag 1973
The Laetaeid Oxygen Debt
in Frogs after One Hour's Apnoea in Air
D a v i d R. J o n e s a n d T a r i q M u s t a f a
Department of Zoology, University of British Columbia, Vancouver
Received April 9, 1973
Summary. 1. During one hour's apnoea in air curarised Rana pipiens showed
a reduction in oxygen metabolism of some 21%. When artificial ventilation was
restarted the frogs consumed 178 r more oxygen in the first 10 rain than in comparable periods before the apnoea. Over the recovery period of one hour the frogs
consumed 350 tzl more oxygen than in the hour before the apnoea.
2. Blood lactic acid concentrations increased during apnoea and fell during the
recovery period, the highest lactate levels being attained at the end of the apnoea
and not during the initial part of the recovery period. The lactate: pyruvate ratio
rose during apnoea. Blood glucose concentrations were above pre-apnoeic values
in the early part of the recovery period.
3. After one hour's apnoea in oxygen, during which there was no significant
reduction in oxygen metabolism, curarised frogs displayed no oxygen debt when
artificial ventilation was restored.
4. The results of the present experiments are discussed in the light of previous
data and it is concluded that, in normal frogs, the increase in oxygen uptake following submergence has three elements, the major one being the oxygen cost of
increased activity in the form of hyperventilation.
Introduction
Curarisation of frogs h a s p r o v e d useful for i n v e s t i g a t i o n of b o t h
m e t a b o l i c a n d c i r c u l a t o r y effects of p r o l o n g e d a p n o e a in air, as well as
allowing a s s e s s m e n t of t h e o x y g e n d e b t in t h e absence of a c t i v i t y
(Jones, 1972b). W h e n artificial v e n t i l a t i o n is r e s t a r t e d a f t e r one h o u r ' s
a p n o e a in air, frogs (Rana esculenta, a v e r a g e w t = 3 7 . 8 g) consume
110-130 ~1 m o r e o x y g e n in t h e first 10 m i n t h a n in c o m p a r a b l e periods
before t h e a p n o e a . This excess u p t a k e is a b o u t equal t o t h e a m o u n t of
o x y g e n c a l c u l a t e d to h a v e been r e m o v e d f r o m t h e o x y g e n store of t h e
blood d u r i n g a p n o e a (Jones, 1972b). H o w e v e r , d u r i n g a r e c o v e r y
p e r i o d of one h o u r m o r e o x y g e n is c o n s u m e d c o m p a r e d w i t h t h e preapnoeic p e r i o d t h a n can be a c c o u n t e d for b y r e p l e n i s h m e n t of o x y g e n
stores. A n a l y s i s of a c i d - b a s e b a l a n c e of a p n o e i c frogs s u g g e s t e d t h a t
m e t a b o l i c acids were being a d d e d t o t h e b l o o d d u r i n g a p n o e a (Jones,
1972b), a n d i t is possible t h a t t h e m o r e p r o l o n g e d increase in o x y g e n
16
D. R. Jones and T. Mustafa:
u p t a k e t h r o u g h o u t the recovery period is used to r e p a y a lactacid
oxygen debt.
Exposure of R. pipiens to 100 % N 2 results i n depletion of v e n t r i c u l a r
a n d gastronemieus muscle a n d of hepatic glycogen c o n c e n t r a t i o n s with
time, a n d iodoaeetate poisoned frogs s u c c u m b i n t o t a l a n o x i a m u c h
sooner t h a n n o r m a l a n i m a l s (Rose a n d D r o t m a n , 1967). This work,
with t h a t of A r m e n t r o u t a n d Rose (1971) i n which blood sugar a n d
lactic acid levels were m e a s u r e d i n Bufo cognatus, gives a clear i n d i c a t i o n
t h a t anaerobiosis provides energy for m a i n t e n a n c e of physiological
i n t e g r i t y i n a n u r a n s d u r i n g t o t a l anoxia, b u t does n o t give a n y idea of
the degree of anaerobiosis which m i g h t be expected to occur u n d e r
conditions such as diving, when oxygen m e t a b o l i s m is m a i n t a i n e d
between 50-67 % of the pre-diving level (Jones, 1967).
C o n s e q u e n t l y i n the present e x p e r i m e n t s a n a t t e m p t has been
made to measure the lactate a n d p y r u v a t e changes u n d e r g o n e b y
curarised frogs (R. pipiens) d u r i n g a n d after one h o u r ' s apnoea i n air
a n d to relate this to the extra oxygen c o n s u m e d d u r i n g recovery from
apnoea.
Methods
The experiments were performed on 40 R. plpiens at 24~ C. The frogs were
obtained from commercial suppliers and acclimated to a temperature of 2 4 • 2~
for a period of not less than 30 days. During the period of acclimation the frogs were
fed mealworms and the individual members of the population suffered no gross
weight changes.
Twenty-three frogs were used to determine oxygen consumption in a respirometer at 24 ~: 0.05~C. Heart rate was also determined in twelve of these animals by means of copper wires sewn under the skin (Jones, 1970). A cannula (2 cm
long and 2--3 mm diameter) was inserted into the tip of one lung under MS 222
Sandoz anaesthesia (300 rag/L), achieved by immersing the animal in the solution.
After recovery from the anaesthesia the frogs were paralysed by injection of a
solution of curare into the dorsal lymph sac (0.02 mg/g wet weight). A complete
description of the experimental procedures and precautions taken to ensure accuracy when using the respirometer has already been given (Jones, 1970, 1972b).
When the frogs were in the respirometer the lungs were ventilated using a variable
displacement pump (Jones, 1970; rate 6-7/min, volume 1 ml/10 g wt) and apnoea
was achieved by switching off the pump with the lungs collapsed. Oxygen consumption and heart rate were measured in 12 frogs before, during, and after one
hour's apnoea in air and in 11 frogs these procedures were repeated in an atmosphere of pure oxygen.
Fifteen frogs were used to measure blood lactic and pyruvic acids. In these
experiments the body temperature was maintained at 2 4 ~ 1~ by placing the
curarised animals, attached to a cork board, on a warming tray. Water at 24~
was added frequently to keep the skin moist. Before curarization a cannula was
inserted into the lungs and a catheter (P. E. 50) into the sciatic artery under MS 222
Sandoz anaesthesia. Blood samples were withdrawn from the sciatic artery before,
during, and after a period of one hour's apnoea in air. The sample size was 50 or
100 tzl depending on the size of the frog. After collection the sample was fixed in
17
Anaerobiosis and the Oxygen Debt
30
I
1300
1200
02 Uptake
(pk/10 min.)
75
70
1100
65
1000
60
9OO
55
Heart Rate
@eats/min~
800
700
6OO
500
40C
i
60
120
1 1 1 1 1
~80
Time (rain.)
Fig. 1. Oxygen consumption and heart rate of curarised frogs before, during and
after one hour's apnoea in air. The artificial ventilation was stopped at 60 min and
restarted at 120 min. Average values of 12 experiments, average weight= 37.1 :[:
1.8 g (n-- 12). o heart rate
perchloric acid (8%), shaken vigorously, and stored at - - 2 0 ~ for later
analysis. The blood samples were analysed for pyruvic and lactic acid concentrations using methods described in Sigma Technical Bulletins No. 726-UV and
826-UV, with appropriate modifications in view of the small sample volumes. On
average replicate analysis of the same sample gave results which did not differ by
more than :~ 3 %. Blood glucose concentrations were measured in some blood
samples taken from the period before and 10 min after the hour's apnoea, using
techniques outlined in Sigma TechnieM Bulletin No. 510.
All results were analysed statistically by Fisher's t-test and 5 % was regarded
as the fiducial limit of significance. When mean values are given in the text and
figures they are presented ~ S. E. ]~I.
Results
a) Oxygen Consumption, be/ore, during, and after One Hour's Apnoea
in Air
The average oxygen c o n s u m p t i o n of the twelve curarised frogs on
artificial v e n t i l a t i o n before the apnoeie period was 782~: 18 ~1/10 min.
D u r i n g apnoea, which was started with the lungs collapsed, oxygen
u p t a k e fell a n d i n the period from 10 to 20 rain after onset of apnoea
it was significantly below the pre-apnoeic value (Fig. 1). Oxygen u p t a k e
stabilized a t this value for the r e m a i n d e r of the apnoeie period (Fig. 1).
2
J. comp. Physiol., Vol. 85
18
D. g. Jones and T. Mustafa:
During the apnoea frogs consumed 983 ~1 less oxygen than in the
hour before and heart rate fell by about 5 beats/rain although this change
was not significant (Fig. 1).
When artificial ventilation was restarted oxygen uptake rose dramatically and was significantly above that observed in the pre-apnoeic
period for the first 10 rain of recovery (Fig. 1). During this time frogs
consumed 960 • 50 ~1 of oxygen, an increase of 178 ~l over that consumed in a comparable pre-apnoeic period. The oxygen uptake remained elevated, compared with the pre-apnoeic levels, throughout
the remainder of the recovery period of one hour, but the difference
was not significant. During the one-hour recovery period the frogs
consumed, on average, 350 ~l more oxygen than they did in the hour
before the apnoea. Heart rate returned to control levels immediately
artificial ventilation was restarted.
b) Blood Lactic and Pyruvic Acid Concentrations be/ore, during,
and alter One Hour's Apnoea in Air
On artificial ventilation before the apnoea average blood lactic
acid concentration was 7.74-t-0.77 raM/1 (Table 1), a figure considerably
above that reported for Bufonidac (Leivestad, 1960; Armcntrout and
Rose, 1971), but closer to reported values for Ranidae (Hashimoto and
Nukata, 1951). On artificial ventilation the blood glucose level (52.5
3 mg %) was higher than that recorded for R. tigrina by Sabnis and
Rangnekar (1968) and Maitrya et al. (1970) but in the range of values
quoted for other Ranidae by Smith (1950, 1954) and Hashimoto and
Nukata (1951).
The concentration of lactic acid in the blood increased during
the period of one hour's apnoca (Table 1). The highest levels were
recorded at the end of the period of apnoca and not during the initial
recovery period (Table 1). Blood lactate decreased during the hour
following restarting artificial ventilation to 8.79 -t- 0.69 raM/1 (Table i).
Due to the marked variability in lactic acid levels between animals none of
the changes were significant when analysed by Fisher's t-test; however
as lactic acid concentrations increased in all animals between the preapnoeic and end of the apnoeic period, and decreased during the recovery
period, the changes in lactate between these periods were significantly
different at the 2.5 and 1% levels respectively when examined by
Wilcoxon's paired data test. Blood glucose levels increased by 5.1 rag-%
between the pre-apnoeic period and after l0 rain recovery from the
apnoea (Table 1).
The lactate: pyruvate ratio increased during the apnoea but returned
to the pre-apnoeic levels during the recovery period (Table 1). Heart
Anaerobiosis and the Oxygen Debt
19
Table I. Lactate, pyruvate and glucose concentrations in arterial blood of eurarised
R. p@iens before, during, and after one hour's al0noea in air. Average values from
determinations on 15 animals, average weight = 30 :k 2.25 g
Pre-apnoea
Lactate (raM/l)
7.74:E 0.77
60 min apnoea 10 min recovery 60 rain recovery
9.92• 1.04
9.75• 1.11
8 . 7 9 i 0.69
Lactate:
pyruvate ratio
12.54-2
16.89-t-2.97
13.194- 1.74
11.06•
Heart rate
(beats/min)
60 ~ 3.5
50 • 3
55 • 3
58 q- 3
--
57.3 -4-4
--
Glucose (rag- % ) 52.2 • 3
r a t e of these animals decreased d u r i n g t h e a p n o e a b y 10 b e a t s / m i n
b u t t h e r a t e a f t e r 60 rain a p n o e a was n o t s i g n i f i c a n t l y different from
t h a t in t h e p r e - a p n o e i e period. H e a r t r a t e rose o n l y when artificial
v e n t i l a t i o n was r e s t a r t e d a n d control levels were n o t a t t a i n e d u n t i l
a f t e r 40 m i n recovery.
c) Oxygen Consumption be~ore, during, and after One Hour's Apnoea
in Oxygen
The a v e r a g e o x y g e n u p t a k e of t h e eleven curarised frogs used in
this series of e x p e r i m e n t s was 9 1 9 : ~ 3 2 ~1/10 m i n in t h e pre-apnoeic
period. H o w e v e r , these a n i m a l s were significantly h e a v i e r t h a n those
used in section (a) so t h a t on a u n i t weight basis t h e difference in
o x y g e n u p t a k e b e t w e e n t h e two groups of frogs was negligible. This
finding is in a g r e e m e n t w i t h t h a t m a d e p r e v i o u s l y on R. ~@iens breathing n o r m a l l y (Jones, 1967); t h a t is, o x y g e n u p t a k e is n o t e l e v a t e d
during exposure to p u r e oxygen. As can be seen h-ore Fig. 2 there
were no significant changes in o x y g e n u p t a k e from the pre-apnoeic
level either during t h e a p n o e a or t h e 40 rain r e c o v e r y period.
Discussion
The p r e s e n t d a t a h a v e definitely e s t a b l i s h e d t h a t R. pipiens can produce energy during a p n o e a in air b y m e a n s of a n a e r o b i c processes. However,
t h e a m o u n t of e n e r g y o b t a i n e d is small c o m p a r e d w i t h t h a t o b t a i n e d from
o x i d a t i v e m e t a b o l i s m during a p n o e a . F o r instance, o x i d a t i v e processes
d u r i n g a p n o e a can c o n t r i b u t e u p to 20.5 calories to t h e energy b u d g e t
in t h i s size of frog, assuming t h a t t h e I~. Q. = 1 as was recorded for
R. esculenta u n d e r similar conditions (Jones, 1972b), whereas the t o t a l
anaerobic c o n t r i b u t i o n is only a b o u t 11% of this value assuming t h a t
l a c t a t e levels in the muscles are t h e same as those in blood a n d t h a t
2*
20
D. R. Jones and T. Mustafa:
VENTILATION ON
1200-
Vs
OFF
VENTILATION ON
1lO0loo09OO800OXYGEN UPTAKE
(,,t / lo m~..)
zoc6oO5OO40030020Oioo 2
o
o
2"
4'
~o
8'
,oo
;2o
'
,:,o
,~o
TME
(m~nu,.~)
Fig. 2. Oxygen consumption of curarised frogs before, during and after one hour's
apnoea in oxygen (Po~ = 760 mm Hg). ArtificiM ventilation was stopped at 60 min
and restarted at 120 min. Average values of 11 experiments, average weight=
48.2 :[: 3 g ( n ~ 11)
these tissues together constitute about 40% of the animal's total
body weight. Huckabee's (1958) basic assumption was t h a t blood
lactate and pyruvate concentrations reflect intramuscular concentrations
in mammals, although this has not been confirmed in all cases (Karlson,
1971). However, the pattern of change in blood lactaeid concentrations
during recovery, being quite unlike that seen in higher vertebrates
(Scholander, 1940), would suggest that Huckabee's (1958) assumption
holds for amphibians under these conditions. The second assumption
made in the calculation of the anaerobic contribution to the apnoeic
energy budget was that the blood and muscles contributed 40 % of the
total body wet weight. Total blood volume is about 8 % of body weight
(Prosser and Weinstein, 1950), whereas careful dissection of the muscles
from two specimens of R. pipiens showed t h a t the muscles contributed
about 30-33 % of the body wet weight.
I n a survey of oxygen uptake b y non-curarised anurans during one
hour's submergence at 17~ it was found that oxygen uptake of R.
Anaerobiosis and the Oxygen Debt
21
pipiens (av. wt. 32 g) was reduced by about one-third from the predive level whereas in R. esculenta (av. wt. = 36 g) the reduction was by
some 50 % (Jones, 1967). The difference between these two species in
regard to cutaneous oxygen uptake has been confirmed by studies
on eurarised animals during apnoea in air. For curarised R. esculenta
(av. wt. = 3 8 g) the reduction in oxygen uptake during one hour's
apnoca in air (Jones, 1972b) was more than twice that observed for
curarised R. pipiens (av. wt. = 37 g). In fact the rather small decrease in
oxidative metabohsm in the present experiments suggests little reduction
in overall energy metabolism during the apnoea, particularly if R.
pipiens exhibits R. Q. changes akin to those of R. esculenta during
apnoea (Jones, 1972b). If this is the case then, assuming the same
calorific equivalents for fat-carbohydrate metabolism commonly given
for man, the pre-apnoeic energy production for R. pipiens in section
(a) is of the order of 22.9 cals/hr. During apnoea oxidative processes
contribute 20.5 cals/hr, to the energy budget and the anaerobic contribution is 2.3 cals/hr [if animals of the weight range used in section (a)
undergo lactate changes similar to those in section (b)]. The slight
bradycardia shown in the present experiments ]ends support to the
conclusion that the energy budget during apnoea was little changed
from that in the pre-apnoeic period (Jones, 1972a). However, this may
not obtain for all a,nurans during periods of solely cutaneous respiration
since both Leivestad (1960) and Jones (1972b) recorded decreases in
heat production of both toads and frogs during submergence and apnoea
in air respectively.
The total oxygen debt displayed by the present animals was about
350 ~1 in the one hour recovery period. The pattern of oxygen uptake
during recovery closely parallels that observed in curarised R. esculenta
(Jones, 1972b) and can probably be explained on the same basis. That
is, the signkficant increase in uptake over the pre-apnoeic level in the
first 10 rain of recovery is probably used to replenish the oxygen store
of the blood and other more minor stores. Judging from the results of
R. esculenta (Jones, 1972b) the amount of oxygen required is in the
range of 120-130 ~1. For R. pipiens this would probably be somewhat
high since it was shown in R. esculenta (Jones, 1972b) that the amount
by which arterial oxygen tension falls is directly proportional to the
reduction in oxygen uptake and the fall in oxygen uptake in R. esculenta,
as has already been pointed out, was by 44% compared with only about
half this value in R. pipiens. However, it must be appreciated that
differences in the shape of the oxygen dissociation curve, the magnitude of the Bohr Effect, the total oxygen capacity of the blood, as well
as the degree of cutaneous vaseularization, may all contribute to substantial differences between the two species in regard to oxygen uptake
during apnoea.
22
D. 1~. Jones and T. Mustafa:
The difference between the oxygen required to replenish stores
and the total excess uptake over the recovery period, in the present
case some 220-230 t~l, probably represents that which is used to repay
the lactacid oxygen debt. Huckabee (1958) introduced the concept of
"excess lactate", which is calculated from simultaneous lactate and
pyruvate concentrations, the assumption being that lactate might be
expected, from the concept of the law of mass action, to be formed due
to an accumulation of pyruvate, and consequently "excess lactate"
would be expected to correlate better with the oxygen deficit. However,
recent studies have indicated that, at least during heavy exercise
in mammals, lactate accumulation is directly related to the oxygen
deficit in exercising muscles (Kar]sson and Saltin, 1970). Furthermore, in the present experiments, at the end of the apnoea, blood
pyruvie acid levels were slightly below the pre-apnoeic values, a result
similar to that in the freshwater turtle after prolonged anaerobiosis
(Robin et al., 1964). In view of these results it seems more reasonable to
calculate the lactacid oxygen debt from the actual measured values of
lactic acid in the blood. Consequently, animals of the weight range
used in section (a), if undergoing lactacid changes similar to those in
section (b), eliminate 3.365 ~M of lactic acid from the blood during the
recovery period which, assuming that 20% is oxidized (Scholander,
1940), will require 45 ~1 of oxygen. Therefore, the total amount of
oxygen required if the blood concentrations parallel those of the muscles
is five times this value since blood and muscles form 40 % of the total
body wet weight. When this value is added to the calculated requirement
to replenish oxygen stores (120 i~l) then the total calculated debt is
345 ~l, a value which is in extremely good agreement with the recorded
value of 350 ~l.
Since the two major components of the post-apnoeie oxygen debt
have now been definitely established (Jones, 1972 b) it might be predicted
that eurarised frogs would exhibit no increase in oxygen consumption after
one hour's apnoca in oxygen, provided that oxygen metabolism is not
changed significantly during th e apnoea. This was in fact confirmed in the
final series of experiments reported in this paper. However, similar experiments performed previously with non-curarised R. pipiens (apnoea being
induced by submergence) gave totally different results in that the
oxygen debt following submergence in water of Po2 = 760 mm Hg was
unchanged from that observed after submergence in aerated water
(Poe = 150 mm Hg), although oxygen metabolism was not reduced under
the former condition (Jones, 1967). In fact the oxygen debt displayed
by normal animals during the first 30 min of recovery from submergence
in oxygenated water was some 5 to 6 times greater than that recorded
in the present animals after one hour's apnoea in air [this calculation
being performed on a unit weight basis and after compensation for the
Anaerobiosis and the Oxygen Debt
23
difference in t e m p e r a t u r e b e t w e e n t h e two series of e x p e r i m e n t s (Rieck
et al., 1960)]. I n view of this t h e suggestion m a d e , a t t h a t time, t h a t t h e
o x y g e n d e b t was m a s k e d b y t h e o x y g e n cost of excess a c t i v i t y in the
form of h y p e r v e n t i l a t i o n a p p e a r s v a l i d (Jones, 1967). I n r e t r o s p e c t
it would a p p e a r t h a t h y p e r v e n t i l a t i o n occuring a f t e r no r e d u c t i o n in
o x y g e n m e t a b o l i s m during submergence is caused b y t h e r e q u i r e m e n t to
eliminate carbon dioxide a c c u m u l a t e d in blood a n d tissues (Lenfant a n d
J o h a n n s e n , 1967; Jones, 1972b). C o n s e q u e n t l y i t can now be p r o p o s e d
t h a t in n o r m a l frogs, during r e c o v e r y from p r o l o n g e d submergence, t h e
increase in o x y g e n u p t a k e a b o v e n o r m a l levels has t h r e e factors: (I) the
r e p l e n i s h m e n t of o x y g e n stores, p a r t i c u l a r l y t h a t of the blood (Jones,
1972b), (II) t h e r e p a y m e n t of t h e l a c t a e i d o x y g e n d e b t (present d a t a )
a n d ( I I I ) t h e provision of m e t a b o l i c energy for excess a c t i v i t y in t h e form
of h y p e r v e n t i l a t i o n (Jones, 1967). The l a s t is b y far the m a j o r contribu t o r to t h e e l e v a t i o n in o x y g e n u p t a k e .
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24
D . R . Jones and T. Mustafa: Anaerobiosis and the Oxygen Debt
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David R. Jones and Tariq Mustafa
Department of Zoology
University of British Columbia
Vancouver 8, B. C., Canada