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1 Integrative Zoology, The aim of this study was to measure the
energetic consequences of hypoxia in different types of skeletal muscle
within a single tilapia species (n = 5). To that aim, 81.0 MHz
31P-nuclear magnetic resonance
(NMR) spectra were collected, alternately, from three surface coils
placed adjacent to the tissues of interest (dorsal white muscle,
ventral white muscle, and lateral red muscle) during a graded hypoxia
load over 6 h followed by a 5-h recovery period. The fish were
contained in a flow cell, enabling us full control of the oxygen
content of the bathing medium. The intracellular pH and the
concentrations of ATP, phosphocreatine (PCr), and
Pi were determined from the NMR
spectra. For normoxia, biochemical differences for
[
in vivo 31P-nuclear magnetic
resonance; intracellular pH; metabolic depression
THE MYOTOME OF MOST TELEOST fish is composed of
distinct regions of red and white muscle. Red muscle (RM) has a
well-developed blood supply, a high myoglobin and mitochondria content,
high concentrations of lipids and cytochromes, and high activities of
respiratory and citric acid cycle enzymes. RM has an active aerobic
metabolism, using both carbohydrates and lipids as substrates (12, 16,
17). The bulk of the muscle tissue in fish, however, consists of white
muscle, which depends mainly on anaerobic glycolysis for its energy
supply. White muscle has a much higher glycolytic potential and a
higher buffer capacity than RM (12, 16, 17). Biochemical differences
between those two tissues were observed for amino acids (38),
phosphorylated compounds (30, 47), and enzyme characteristics (3, 48).
Due to the clear differences between red and white muscle, a different
response to a stressor, such as a graded hypoxia load, might be
expected in these two types of tissue.
31P-nuclear magnetic resonance
(NMR) is widely used to study the biochemical effects of environmental
changes on animal tissues in vivo (34). The effects of hypoxia and
anoxia on white muscle tissue have been investigated in unanesthetized
fish in a specially developed flow-through cell (31). Spectra were
obtained with good resolution and high signal-to-noise ratio. These
were indicative of a high phosphocreatine
(PCr)-to-Pi ratio (>25-40)
and neutral tissue pH (7.1-7.3) under control conditions. Exposure
to anoxia resulted clearly in the activation of anaerobic processes, in a decline of high-energy phosphates, and in metabolic acidosis (36,
49). Two important conclusions were drawn from these studies:
1) the decline of [PCr]
appeared to be coupled to acidosis (35, 49) and
2) there were characteristic
differences between species (i.e., goldfish showed a strongly reduced
acidosis compared with carp, whereas tilapia exhibited an intermediate
response). It was concluded that these differences were related to the
occurrence of metabolic depression, which can be defined as a
depression of the metabolic rate below the standard metabolic rate
(SMR). This metabolic depression was observed in goldfish, but not in carp (43).
There is a marked difference between anoxia and hypoxia. Under hypoxic
conditions, aerobic and anaerobic energy production may work
simultaneously. In former studies, NMR experiments were performed with
the same setup measuring with a surface coil. In these studies only
dorsal white muscle (DWM) was measured under controlled conditions in
which oxygen levels were declining stepwise. These studies showed no
major differences between species: goldfish, tilapia, and carp (44).
All three of these species demonstrated rapidly declining energy stores
and high glycolytic activities at levels <10% air saturation. In
addition, there was no indication of metabolic depression.
So far, in vivo NMR studies have not differentiated between the
different locations and different muscle types within the same animal.
Metabolic activity in an animal tissue may be related to its location
and its use. Lateral RM is used continuously for slow swimming and
steady-state movements, whereas dorsal white musculature is primarily
used above certain swimming speeds. Therefore, under hypoxic
conditions, the metabolic activity of RM is expected to be much higher
than that of white muscle. For these reasons, we hypothesize that
stabilization of the energy state of RM, under hypoxic conditions, is
more important for the animal than that of white muscle. So, in this
study, we aimed for simultaneous measurement of the response to
hypoxic stress in white muscle and RM. Two different regions of
white muscle were compared to test the homogeneity of the response
within the same tissue type. To assess the tissue-specific response,
three separate 31P-NMR surface
coils were placed at appropriate locations. To avoid significant
interference between the coils, we used fish that were larger than
those used in previous studies. Although in earlier studies we worked
with 60- to 80-g fish in an 8-cm bore magnet (31, 36, 37, 44, 45, 49),
in this study we used fish of 400 g in a 40-cm bore magnet. The data
indicate that, for normoxia, biochemical differences existed between
the red and white muscle, with respect to the concentrations of
Animals and Handling
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
-ATP], [PCr], and [sugar
phosphates] (SP) were observed between all three locations,
especially between the red and white muscle. During hypoxia stress,
loss of phosphorylated compounds
(PCr+Pi+SP) was observed at all
locations but was the most severe in red muscle. When the aerobic
(respirometry) and anaerobic
(31P-NMR) ATP production via an
energy balance are compared, flexible metabolic depression is
demonstrated during anaerobioses. It is concluded that control of the
aerobic and anaerobic component of metabolism during metabolic
depression is independent of each other.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
-ATP, PCr, and sugar phosphates (SP). Furthermore, during hypoxia
stress, loss of phosphorylated compounds
(PCr+Pi+SP) was observed at all
locations, but was the most severe in RM. An energy balance was drawn
from NMR data and compared with aerobic oxygen consumption. Comparison
of the anaerobic response [PCr depletion and drop in
intracellular pH (pHi)]
with the aerobic response (oxygen consumption) showed that the
metabolic rate dropped below the standard metabolic rate (SMR), a
process called metabolic depression.
MATERIAL AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
NMR Measurements
31P-NMR studies were performed at 81 MHz on a SIS (Palo Alto, CA) 200/400 in vivo NMR spectrometer interfaced to a 40-cm horizontal 4.7-Tesla Oxford magnet. Three different radio frequency (RF) coils were used for signal acquisition, alternating from coil to coil in a cyclic protocol (see below). The resonance frequency of each coil was brought close to the 31P frequency by placing appropriate tuning capacitors close to the coil. The loading conditions during the in vivo experiments were simulated with an isotonic saline solution. The placement of the coils, their diameter, and the number of turns were as follows: RM, diameter 1.4 cm, two turns; ventral white muscle (VWM), diameter 2.5 cm, two turns; and DWM, diameter 4.0 cm, single turn. They were fixed to the flow cell at positions corresponding to 6.5 cm between the first and the second coil, 4.75 cm between the first and the third coil, and 7 cm between the second and the third coil (Fig. 1). In pilot experiments, it was observed that RM of tilapia of this size (~400 g) had a diameter of ~6 mm. Because the radius of the RM coil was 7 mm, it can be concluded that the tissue sampled by the RM coil consisted of a minimum of 86% of RM. After the surface coils had been properly placed, the flow cell was positioned in the magnet such that the largest coil was in the isocenter of the magnet. Thereafter, the coils were individually tuned to the 31P Lamor frequency using three separate tuning/matching circuits. Interference between the coils was minimized by short circuiting at the RF shield of the magnet by placing end caps on the coils that were not used. Extensive tests demonstrated that coupling between the coils was negligible. After being tuned and matched to the 31P frequency, the magnetic field was optimized for each coil by a pulsation on the H-1 frequency and the use of the free-induction decay (FID) signal from water for shimming. Water line widths were typically between 32 and 64 Hz. Next, the 31P-NMR protocol was started. For each coil, excitation was with a 1-ms adiabatic half-passage pulse with offset at the PCr peak. The optimal pulse power for the adiabatic regimen was optimized once and subsequently kept constant for each experiment. The rationale for using adiabatic half-passage pulses for signal excitation was that the tip angle is exactly 90° throughout the sensitive volume of the coil. A potential disadvantage of adiabatic RF pulses is that their off-resonance performance is less good than that of hard pulses. A consequence of this may be, for example, that the signal intensity of the
- to the -peak of ATP is below one. Nevertheless, we
considered the use of adiabatic pulses advantageous because of their
uniform excitation profile, in spatial terms, and their immunity to
power variations.
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Control experiments on an in vitro sample containing PCr and ATP in a
4:1 molar ratio were performed to assess the reliability of the
three-coil setup in terms of the PCr-to-ATP ratio determined from the
spectra. The solution was contained in a plastic bag that was placed in
the flow cell. The coils were positioned as in the above in vivo
studies. It appeared that similar metabolite ratios were measured with
each coil, both when present simultaneously and when tested
individually with only one coil present. Importantly, the ratio of
-ATP to PCr was identical when adiabatic pulses were used in the
above setup and when hard pulses were used with a small vial placed
centrally in a homogeneous solenoidal
31P coil. The mean line broadening
in five experiments was 59.4 Hz for the first coil, 54.8 Hz
for the second coil, and 56.8 Hz for the third coil. For each
FID, 4,096 complex data points were accumulated for an
acquisition time of 0.41 s and spectral width of 5,000 Hz. A repetition
time of 10 s was used. For coil
2 (DWM), a series of nine spectra (24 scans each) was collected, whereas for
coils 1 (RM) and
3 (VWM), 36 scans were averaged for a
single FID for each experimental condition.
The pHi was estimated from the
difference in chemical shift between PCr and
Pi. The pH measurements were
calibrated using several model solutions, as described previously (36).
The pHi was calculated using the
formula
![pH<SUB>i</SUB> = 6.72 + log [(&sfgr; − 3.27)/(5.69 − &sfgr;)]](/content/vol277/issue5/fulltext/R1501/img001.gif)
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corresponds to the chemical shift between PCr and
Pi.
ATP concentrations in extracts (see below) were measured with the enzymatic assay of Lamprecht and Trautschold (19). The mean ATP concentration was 5.03 ± 0.57 µmol/g in white muscle and 3.60 ± 0.40 µmol/g in RM. [Total creatine] was measured according to the methods of Ennor and Stocken (10). A [total creatine] of 32.26 ± 1.38 µmol/g wet wt was found in white muscle and 23.80 ± 1.19 µmol/g wet wt in RM.
[PCr] for white muscle and RM was estimated from the ratio of the relative resonance intensities (RRIs) of PCr and ATP, starting from a normoxic RRI for ATP. This corresponds to a tissue concentration of 5.03 µmol/g wet wt in white muscle and 3.60 µmol/g wet wt in RM.
Protocol
The fish used in the in vivo 31P-NMR experiments were exposed, for a period of 60 min, to normoxic conditions [100% air saturation (AS)]. This was followed by a stepwise decrease in oxygen content to 40, 30, 20, 10, 5, and 3% AS for a period of 60 min each. Hypoxia was followed by a variable period of anoxia [method described by van Ginneken et al. (44)]. Thereafter, the animals were exposed to a recovery period of 5 h at 100% AS. Surface coils 1, 2, and 3 were placed on the caudal part of the lateral RM, the rostral part of the epaxial white muscle, and the VWM next to the gill, respectively (Fig. 1). Over each 60-min period, 11 spectra were collected in the following sequence: 9 spectra from coil 2 (24 scans each), 1 spectrum from coil 1 (36 scans), and 1 spectrum from coil 3 (36 scans). During anoxia, because of the limited period, the measurements were made with coil 2 only. The anoxia exposure length was based on the degree of exhaustion of the PCr stores. When the PCr peak was depleted until its peak height equalled the-ATP peak,
reoxygenation was activated. Pilot experiments revealed that mortality
occurred only when PCr levels were reduced even further. In the
experimental group, no mortality was observed during or after the experiments.
Oxygen Registration
Two types of respirometric studies were performed: 1) directly in the flow-through cell in the NMR apparatus with an immobilized fish (see Table 1) and 2) in parallel experiments in a respirometer with free-swimming fish to determine the SMR and percentage of metabolic depression during a graded hypoxia load (see Table 2).Respirometry in the flow-through cell. The oxygen content of the medium was measured with a registration system [described by van Ginneken et al. (44)] that consisted of a computer-driven rotating valve (Bürckert type 332-E-B-G1/4-220/50-F-024), which alternately directed the waterflow from the inlet or outlet of the flow-through cell over an oxygen electrode (Radiometer Copenhagen E5046 with thermostatted cell D616). The oxygen detection system was a Radiometer Denmark Digital Oxygen analyzer PHM 72 with a PO2 module PHA 932. The oxygen electrode was calibrated in a 10% sodium sulfite solution (zero) and at 100% in air-saturated water. In the reference position, normoxic or hypoxic water from the gas equilibration cylinders flowed directly over the electrode. In the measurement position, the water first passed through the flow-through cell with fish and then over the electrode. The selected time intervals for the two different valve positions were 10 min in the reference position and 50 min in the measurement position.
The oxygen consumption was calculated as
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Metabolic rate measurements in a respirometer. Seven fish were placed individually in a flow-through respirometer of 20 liters at normal oxygen levels (80% AS; Ref. 33). The supply of water to the respirometer was regulated by an EIL O2 monitor/controller type 9401. The controller activates a solenoid valve when the PO2 value falls below a set point. Air saturated water flows from a storage tank into the respirometer chamber until the preset level is reached. The flow is measured by a digital flowmeter, which is coupled to a counter and data logger (33). Temperature in the experimental set up was kept at 20 ± 0.1°C.
Sampling Procedure and Tissue Preparation
Tissue samples were taken in parallel experiments to measure ATP and creatine levels. Fish were acclimatized to the experimental set up at 20°C for 2 days under normoxic conditions to reduce handling stress. Fish (anesthetized with 200 ppm MS-222) were killed by decapitation. Tissue samples of white muscle (within 15 s) and RM (within 30 s) were removed and freeze-clamped between aluminum tongs cooled in liquid nitrogen. The freeze-clamped muscle samples were stored in liquid nitrogen until analysis. Tissue extraction was carried out according to van den Thillart and colleagues (29, 33). Frozen tissue was powdered [in a porcelain tissue grinder (Retsch, type RMO), cooled with liquid nitrogen] together with a 4.0-ml vol of perchloric acid (8% vol/vol) in ethanol (40% vol/vol) containing 4 mM NaF and 10 mM EDTA. The powder was stored for 10 min at
20°C
in a centrifuge tube. Thereafter, the powder was further homogenized on
ice with a high-speed mixer (Salm and Kip BV, type X 1020, Dottingen,
Germany). The homogenate was stored for 30 min on ice and was then
centrifuged (Sorvall RC-5B) for 20 min at 30,000 g. The supernatant was neutralized to
pH 7.0 with 3 M potassium carbonate in 0.5 M triethanolamine. Finally, the extracts were again centrifuged (20 min at 30,000 g), aliquotted into Eppendorf tubes,
and stored at 180°C until use.
The method used for determining the buffer capacity of tilapia red and white muscle was slightly modified after Castellini and Somero (4). Muscle was excised from tilapia after anesthesia with 200 ppm MS-222. The muscle was frozen in 0.5-g parts and powdered in liquid nitrogen together with 10 ml 0.9% NaCl (4.00 meq H2SO4/l). The powder was transferred into a 50-ml titration vial and rinsed with 2 ml H2O. The titration vial was temperature controlled at 20°C. The initial pH of RM was 4.5 ± 0.3 and of white muscle 5.1 ± 0.2. The tissue homogenate was titrated with NaOH (10.00 meq/l) with a Metrohm titrator in steps of 0.200 ml. The pH was read 20 s after addition, which was sufficient to reach a stable pH. The buffer capacity was calculated from the pH increase between 6 and 6.8, which gave, in that part, a linear relationship. For acidification and titration, titrisol grade solutions of 1.000 eq/l were used (Merck).
Method for Estimation of Percentage of Metabolic Depression
Estimation of the percentage of metabolic depression can be inferred by comparing the SMR (see Table 2) with the estimated anaerobic and aerobic ATP production (6). In Teleostei, muscle tissue is, quantitatively, the major metabolically active tissue. It determines, to a great extent, the metabolic rate. For fish, the somatic index of muscle tissue was estimated to be 42%, whereas the remaining metabolically active tissue was estimated to be 21.3% [liver 1.3%, blood 5%, remaining metabolic active tissue (i.e., heart, brain, and gut) 15% (6, 39)]. Because muscle tissue mainly determines the metabolic rate, an energy budget can be estimated on the basis of 31P-NMR data and whole body oxygen consumption. Estimation of the anaerobic ATP production is based on the changes in the most important energy-rich compounds, such as PCr and ATP, and on lactic acid production. However, because the [ATP] was unchanged during the stepwise hypoxia load, only the changes in PCr were considered. Anaerobic lactic acid production, derived from NMR data, was based on the method described by van den Thillart and van Waarde (35). This is based on the formula of Kushmerick and Meyer (18)
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1 · pH
unit1 for white muscle (see
RESULTS), and
b is the stochiometric coefficient of
the PCr hydrolysis reaction, which is pH dependent and corresponds to a
tissue buffer capacity of 0.42 eq/mol over this pH range (14). From the
buffer capacity for fish muscle, and via the 31P-NMR measured 
pH between the
different hypoxia levels, the acid production can be calculated in
milliequivalents per kilogram. From the total acid production and the
buffer capacity, the total lactic acid production can be calculated.
For the anaerobic ATP, 1 mol of lactate corresponds to 1.5 mol ATP
(6). The PCr, used as a buffer for stabilization of the ATP
pool, is measured via 31P-NMR. One
mole of PCr is equivalent to 1 mol of ATP (6). Because we measured
three different tissues, the somatic indexes between the different
tissues (RM, DWM, and VWM) are estimated to be 10, 70, and 20%,
respectively, whereas the relative weight of muscle tissue in fish
corresponds to 42% (39). The level of high-energy phosphates in the
remaining tissues is a minor part of the energy budget, and estimation
during a similar graded hypoxia load is based on HPLC measurements
performed earlier, resulting in an anaerobic ATP production at 30, 20, 10, 5, and 3% of, respectively, 0.47, 0.36, 0.79, 1.27, and 0.78 µmol
ATP · g1 · h1
(39). The combination of muscle tissue and remaining tissue gives a
total anaerobic ATP production, as revealed in Table 6. Because the
oxygen consumption was measured, it was possible to calculate the
aerobic ATP production. This is based on the assumption that 32 mg
O2 correspond to 6 mmol ATP (26).
The sum of the aerobic and anaerobic ATP production gives the total ATP production.
Statistics
For each muscle type, the values for the different parameters (PCr, SP, Pi, pHi, ATP) during the graded hypoxic levels and recovery were compared with the initial 60-min normoxic period. Statistics were performed in SAS (statistical analyzing software). We applied a one-way ANOVA, comparing hypoxia and recovery with normoxia. P
0.05 was considered statistically significant. Normality of the data and
homogeneity of variances were checked by Kolmogorov-Smirnov and
Fmax tests. For
each experiment, the mean value of the metabolites over the
corresponding interval was calculated. Experimental data are presented
as the means ± SD.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The mean oxygen consumption under control conditions and during a
graded hypoxia load and reoxygenation is given in Table 1. The oxygen consumption fell from 16.63 mg · 100 g1 · h1at
normoxia to 2.13 mg · 100 g1 · h1
at 3% AS. Because the standard metabolic rate for tilapia is ~9.78
mg O2 · 100 g1 · h1
(Table 2), the animals were apparently
below this level at 20, 10, 5, and 3% AS. The animals must have
compensated for this by anaerobic processes or by depressing their
overall metabolic rate by ~78%.
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In Fig. 2, a series of
31P-NMR spectra from a typical
experiment for the lateral RM (Fig.
2A), the DWM (Fig.
2B), and the VWM (Fig.
2C) are shown. The patterns are
rather similar: unchanged ATP levels; a drop of PCr during hypoxia,
with a concomitant rise of Pi; and
a rise of SP. Some differences are obvious: the PCr peak in the DWM is
higher than in the RM. Furthermore, the initial [Pi] in the RM does
not approach the high levels observed in dorsal and VWM.
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Data obtained from five complete series, each with three coils and
measured under 13 conditions, are presented in Tables
3, 4, and
5. The 13 conditions are indicated as:
normoxia; hypoxia at 40, 30, 20, 10, 5, and 3% AS; anoxia; and
recovery over five consecutive periods (R1, R2, R3, R4, and
R5). Each condition lasted 60 min, except for the anoxia
period, which was variable (see above).
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Table 3 shows the content of PCr and ATP at the three different locations under the different conditions tested. Enzymatic measurements showed that ATP amounted to 3.60 and 5.03 µmol/g in RM and DWM, respectively. The ATP levels remained constant during hypoxia. In contrast, major changes occurred with the PCr levels. The values of PCr during normoxia were 23.80 mM in the RM, 32.26 mM in the DWM, and 23.77 mM in the VWM. During the 3% AS period, the PCr levels were reduced to 44.8, 50.4, and 56.5% of control in RM, DWM, and VWM, respectively. In RM and DWM, the decline was significantly different, but not in VWM. During anoxia, the PCr level in the DWM fell to 36%. During the first 2 h of recovery, PCr resynthesis proceeded relatively fast in all three tissues.
Table 4 shows the content of SP and Pi in the three tissues. Initial normoxic SP values were 0.85 in RM, 1.53 in DWM, and 1.32 mM in VWM. During the graded hypoxia load, a value of 2.66 mM was reached in RM during 3% AS, whereas this value was 4.02 in DWM and 3.18 mM in VWM. Interestingly, the SP level in the DWM was significantly lower at 40, 30, and 20% AS. During anoxia, the SP rose in DWM to 4.58 mM. During the 5-h recovery period, the SPs were reduced to levels below the initial normoxic values in all three tissues.
Pi values in resting normoxic animals were the lowest in RM (0.87 mM), whereas in DWM and VWM these were 2.37 and 3.0 mM, respectively (Table 4). During hypoxia, Pi values increased in RM, DWM, and VWM to 8.32, 6.21, and 9.08 mM, respectively. During anoxia, the Pi increased further in DWM to 8.43 mM. The highest value of 11.73 mM was reached after 1 h recovery in the DWM. Recovery was fast. Within 3 h, control values were reached in DWM and VWM. In RM, no further recovery of Pi occurred after 3 h.
Table 5 shows the sum of PCr, SP, and Pi, as well as the pHi, of the three muscle tissues during hypoxia and recovery. The sum of PCr, SP, and Pi appeared relatively stable in DWM and VWM, although there was a tendency toward a decline during hypoxia and an increase to normoxic values during recovery. During anoxia, the sum was significantly different in the DWM. Remarkably, the sum declined in the RM during exposure to 20 and 10% AS and did not decrease further during hypoxia, nor did it increase again during 5 h recovery. This resulted in a significant semipermanent loss of 18% of the sum of PCr, SP, and Pi in RM compared with the normoxic situation.
During normoxia, the pHi of RM, DWM, and VWM was 7.24, 7.16, and 7.42, respectively (Table 5). During hypoxia, there is a tendency to acidosis in all tissues, which was significant in RM at 5 and 3% AS and in DWM during anoxia. In the DWM, the pH decreased further during anoxia and reached its lowest level after 2 h of recovery. In the RM, the pattern was similar to that of the DWM; the pH fell from 7.2 at normoxia to 6.8 at 3% AS. The lowest pH value of 6.6, however, occurred after 1 h of recovery. Recovery of pHi in RM, DWM, and VWM took ~5 h.
Table 6 shows the anaerobic ATP
production calculated per hypoxia condition for all
three muscle types. In the calculation we used in this study, measured
buffer capacity for RM was 28.1 ± 3.4 meq · g1 · pH
unit1 and for white muscle
was 40.0 ± 1.4 meq · g1 · pH
unit1.
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Estimation of the percentage of metabolic depression is given in Table
7. It can be seen that the total ATP
turnover fell below the SMR at 3% AS, which is compensated by a
reduction of the metabolic rate until 58% of the SMR.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Oxygen Consumption
The normoxic oxygen consumption rate of the Mozambique tilapia in this 31P-NMR study corresponded to 16.63 mg O2 · 100 · g1 · h1.
In a parallel respirometric study, a standard metabolic rate of
9.78 mg O2 · 100 g1 · h1
for tilapia at 20°C was reported (Table 2). Thus the results presented here indicate that the measured
O2 consumption of the fish in the
NMR flow cell is ~1.70 times the SMR. Even under resting conditions,
some changes in activity level can be expected due to muscle tension
and small movements. Hypoxia exposure depresses the activity level of
free-swimming fish and thus reduces the mean oxygen consumption level.
Only at very low oxygen levels does the oxygen consumption fall
(sometimes) below the SMR (28). This is likely due to a reduction of
muscle tension and blood flow. Although the oxygen consumption in this
study is sampled at relatively long intervals, and therefore activity
peaks cannot be recognized, a similar pattern is obvious. First, we see
a decline in the metabolic rate at 20% AS, but then the oxygen
consumption decreases further to even 21.8% of the SMR at 3% AS.
Evidence for metabolic depression in tilapia has been described before (40, 41, 49). To prove that an animal uses the survival strategy of
metabolic depression, it is necessary to show that the reduction in
aerobic energy production is only partly compensated for by anaerobic
processes. In this study, we cannot quantify the total anaerobic energy
production, but a semiquantitative approach is possible (see Table 6).
SPs
In tilapia, SPs rise to much higher levels under hypoxia than in other fish species, such as common carp and goldfish (44). This indicates, in tilapia, that glycogenolysis, and thus glycogenphosphorylase, is relatively insensitive to feedback inhibition by glucose 6-phosphate. This shifts the control of the glycolytic rate mainly to phosphofructokinase.It is evident from Fig. 2 and Table 4 that the SPs rise in the early phase of hypoxia, before the onset of metabolic acidosis. During anoxia, when the glycolytic rate is high, the SP level does not decrease. This suggests a sufficient capacity of the glycogenphosphorylase reaction.
PCr-to-Cr Ratio
The PCr level appears to differ markedly between the different sample points for RM, DWM, and VWM, respectively, 23.8, 32.3, and 23.8 mM. With in vivo 31P-NMR methods, PCr levels of 24.4 mM have been observed in epaxial white muscle of unanesthetized goldfish (37), whereas in another 31P-NMR study with tilapia, PCr values between 15.8 and 21.0 mM were reported for the DWM (45). The higher values for the DWM in this study are likely related to the weight of the animal. Larger fish usually have higher glycolytic potentials than smaller ones (3).In general, if we compare enzymatic determinations in perchloric acid extracts (obtained via freeze-clamping) with in vivo 31P-NMR measurements, a discrepancy can be observed for the [PCr]-to-[PCr+Cr] ratio (amount of phosphorylated total creatine). With traditional freeze-clamping, it is observed that only a small fraction of the total Cr is phosphorylated. In dogfish muscle, 19% phosphorylation was observed (36.6 µmol Cr/g vs. 8.8 µmol PCr/g; Ref. 2). In white muscle of goldfish, phosphorylation percentage was 27% (21.8 µmol Cr/g vs. 8.3 µmol PCr; Ref. 30). In white muscle of rainbow trout, 45% phosphorylation was measured (25.7 µmol Cr/g vs. 20.8 µmol PCr; Ref. 9). In another study with white muscle of rainbow trout, 39% phosphorylation was measured (27.5 µmol Cr/g vs. 17.5 µmol PCr; Ref. 7).
Only in 31P-NMR studies were very high levels of phosphorylation observed in intact unanesthetized goldfish, 95.3% (1.2 µmol Cr/g vs. 24.4 µmol PCr; Ref. 37). From the results presented in this paper, we calculated the percentage of phosphorylation in DWM as 100% (0 µmol Cr/g vs. 32.26 µmol PCr); in VWM, 73.7% (8.5 µmol Cr/g vs. 23.77 µmol PCr), and in RM, 92.4% (1.96 µmol Cr/g vs. 23.80 µmol PCr). The discrepancy between PCr-to-Cr ratios obtained by in vivo 31P-NMR and conventional freeze-clamping techniques is likely due to handling and artifacts developed during tissue excision and tissue extraction (37, 49).
Pi
The
(PCr+Pi+SP) showed a drop
in red and white muscle during reoxygenation. This decreased phosphate
content may be explained by washout of
Pi or by deposition of calcium
phosphate in mitochondria, which are NMR invisible (24). The former,
however, seems more likely, because the phosphate loss is more
prominent in the better perfused RM. Furthermore, it should be
mentioned that the possibility of a mitochondrial accumulation of
phosphate in primarily RM is also increased due to a probably higher
mitochondrial density in red than in white muscle.
The observed Pi values for tilapia white muscle were between 2.4 and 3.0 mM and in agreement with a previous study (44). Pi has several functions in the cell. First, it may act as a buffer (22); second, it may act as a substrate in glucogenolysis; and, third, it may act as a metabolic regulator (44). A qualification for a metabolic regulator is that the concentration of the compound must be in the range of the Michaelis-Menten constant (Km). In vitro measurement of Pi with isolated mitochondria have shown a Km of 1 mM. In this study, the observed Pi during normoxia (0.9-3.0 mM) is higher than the Km of 1 mM. This indicates that Pi does not function as a metabolic regulator. This observation corroborates that of the 31P-NMR study of Chance et al. (5), in which the [Pi] of isolated mitochondria (state 4) were >1 mM. Only in the liver [where the creatine kinase (CK) equilibrium reaction is not present] is there some evidence that Pi is a regulator of oxidative phosphorylation (27).
pHi
The pHi is the lowest in RM. pHi is 6.63, whereas it is 6.70 in DWM and 6.85 in VWM. This suggests that RM accumulates more lactic acid than white muscle during anaerobioses. Another possibility is that RM has a much lower buffer capacity than, for example, white muscle. This is confirmed by our data where a buffer capacity of RM was recorded of 28.1 ± 3.4 meq · g1 · pH
unit1 and for white muscle
of 40.0 ± 1.4 meq.g1 · pH
unit1. With respect to
recovery, a faster recovery is observed in RM during reoxygenation.
This can probably be explained by lactic acid recycling, which can
occur in RM (15). Lactate dehydrogenase is bifunctional in RM, in
contrast to white muscle, which essentially can only convert pyruvate
to lactic acid (15). Also, in another 31P-NMR study, it was demonstrated
that after ischemia, the
pHi in RM dropped from 7.5 to 5.8 after excision. This drop was only from 7.3 to 6.4 in white muscle
(37).
With respect to the reported pHi values in muscle in this study, it should be noted that the existence of an active H+ transport across the sacrolemma (8, 45) may exist. This probably may lead to an underestimation of the anaerobic metabolism.
A characteristic of oxidative phosphorylation is that no net protons
are formed (14, 22). Consequently, the acidosis observed in this study
during hypoxia and the period of recovery is the net result of
1) the flux through the CK reaction,
2) ion exchange between
intracellular and extracellular compartments,
3) net hydrolysis of adenine
nucelotides (14), and 4) the
glycogen/glucose-to-lactic acid conversion. The drop in
pHi during the first hour of
reoxygenation can probably be explained by the first mechanism, the
flux through the CK reaction. In DWM, the
pHi dropped from 6.85 at anoxia to 6.70 at the first hour of reoxygenation and even to 6.63 during the
second hour of reoxygenation. Most likely, the observed acidosis can be
ascribed to resynthesis of PCr during the initial reoxygenation period.
Resynthesis of PCr is an acidic process, which can be observed from the
CK reaction (35). Also, in the muscle contraction study of Meyer et al.
(20), recovery acidosis has been described. In that study, a
significant decline from pH 6.9 to 6.6 was observed after stimulation
of the rat gastrocnemius muscle. The second process, acidosis due to
ion exchange, is not likely to happen in muscle tissue. It may work the
other way around, eventually, by the
Na+/H+
exchanger, which can be driven by the
Na+ gradient over the plasma
membrane. An active acid extrusion mechanism has been described in
cardiac muscle and seems to rely primarily on both
Na+/H+
exchange and
Na+-HCO3
cotransport (8, 45). The third process, net hydrolysis of adenine
nucleotides is unimportant for acid-base balance in this case, because
the ATP concentration remains constant. The fourth process,
glycogen/glucose-to-lactic acid conversion, however, becomes important
when anaerobic metabolism is activated. It is possible to estimate the
total amount of lactate produced in muscle tissue from the
[PCr] decline and the buffer capacity (35). Thus we find
for DWM, VWM, and RM calculated lactate values of, respectively, 17.24, 27.14, and 17.18 mM over the total hypoxia protocol (see Table 6).
However, because the hypoxic conditions lasted many hours and the RM is
well perfused, the lactate actually produced in the different tissues
may have been much higher.
From two former 31P-NMR studies, an anoxia study (49) and a hypoxia study (44), we find similar values for the lowest pH that can be observed during anaerobiosis in fish muscle. During conditions of anoxia, pH values were 6.9, 6.7, and 6.7 for goldfish, tilapia, and carp, respectively (49). However, during severe hypoxia of 3% AS in white muscle, values of 6.9, 6.8, and 6.8 were reached for these three species. In this study, the lowest pH in DWM was 6.63 during the second hour of reoxygenation, whereas the lowest value during anoxia was 6.85.
High-Energy Phosphates
The concentrations of PCr and ATP in white muscle and RM show striking differences. In general, [PCr] and [ATP] are lower in RM. This was also observed with 31P-NMR techniques in excised goldfish muscle tissue (37) and with HPLC techniques on freeze-clamped tissue of common carp and rainbow trout (47). In ischemic white goldfish muscle at t = 0, a relative resonance frequency for PCr and ATP was observed to be 2.28 and 0.53, respectively. For PCr and ATP in the corresponding RM, this was 1.07 and 0.25. So, on the basis of these observations, it can be concluded that the [PCr] and [ATP] pool in RM is 46.9 and 47.2%, respectively (37). In normoxic common carp and rainbow trout, the [PCr] in RM was found to be 17.8 ± 2.72 and 17.11 ± 2.92 mM, respectively. The [PCr] level in the white muscle of both species was 24.9 ± 1.32 and 20.8 ± 0.91 mM, respectively (47). So, clearly the PCr content of RM is lower than that of white muscle. The same phenomenon is observed with respect to ATP levels. In normoxic common carp and rainbow trout, ATP concentrations in RM are 4.01 and 4.02 mM, respectively, whereas those in white muscle of both species are 5.87 and 6.02 mM, respectively (47). Lower levels of PCr and ATP in RM, compared with white muscle, were also observed in rainbow trout (25), sea bass (21), cod (11), and goldfish (30).Metabolic Depression
In a former study with smaller animals of ~60-80 g, a decrease of the [ATP] to 80% was observed during severe hypoxia (3% AS). During a 6-h recovery period, the [ATP] remained at approximately the same value (44). In this study, the ATP pool remained rather unaffected during hypoxia, even by a consecutive anoxia period. This is likely due to the size of the animals; larger animals have a relatively lower metabolic rate (23). When comparing the depletion rate of PCr and the slope of the pHi decline during hypoxia and anoxia, it is obvious that these processes are slower during anoxia than during hypoxia. Thus the decline in pHi in DWM during severe hypoxia is 0.13 pH units/h, which was significantly different from the decline during anoxia, which corresponded to 0.05 pH units/h. This is remarkable, because under hypoxic conditions the animals were also consuming oxygen. So, energy production under hypoxic conditions is higher than under anoxic conditions, confirming the hypothesis of flexible metabolic depression. The level of metabolic depression is dependent on the severity of the environmental stressor (40, 42). So, we can conclude that in these experiments, tilapia switch over to metabolic depression to survive the severe hypoxic conditions. Metabolic depression is observed among a range of individuals in nature, such as insects in diapause, hamsters during hibernation, diving turtles, and in some fish species (28, 29, 35, 39, 40-43, 46, 49, 50). Just like metabolism can be activated during extreme exercise, metabolism also can be directed oppositely under adverse environmental conditions. The process of metabolic depression was, for the first time, demonstrated directly for anoxic goldfish (42, 46, 50) and hypoxic tilapia (40, 41) using a specially developed 1-liter flow-through microcalorimeter. With this calorimeter, the examiner is able to measure under constant environmental stress-free conditions (1). In tilapia, the metabolic rate in this study (Table 7) was reduced to ~50% of the SMR, whereas during anoxia, goldfish could reduce their metabolic rate to even 30% of SMR. With the use of deconvolution techniques, it was demonstrated that the process of metabolic depression took place on a time scale of minutes (46). For teleosts, this is the first work that describes the response of high-energy phosphates and pHi at different muscle tissues in the same animal with in vivo 31P-NMR during a graded hypoxia load. With this information, an energy balance was calculated, estimating the aerobic and anaerobic ATP production in tilapia. Comparison with the SMR revealed that the environmental stressor hypoxia is not totally compensated for by an activation of anaerobic metabolism in tilapia, but that metabolic depression is a prerequisite for survival in this species.Perspectives
Via two approaches, flexible metabolic depression is demonstrated in this study during anaerobioses: on one hand, by measuring the metabolic rate via respirometry based on the oxygen consumption and, on the other, by depletion of energy stores and accumulation of end products in muscle. In this study, it is demonstrated that during anaerobioses the anaerobic component remains depressed, dependent on the level of oxygen availability. In contrast the aerobic component adjusts itself dependent on the level of oxygen available. These results indicate that the control of the aerobic and anaerobic components is rather independent of each other. Similar results were observed with direct calorimetry and respirometry in two other fish species [goldfish (42, 50) and small tilapia (40, 41)] and two invertebrates, the worm Sipunculus nudus (13) and the bivalve Scapharca inaequivalvis (32). So flexible metabolic depression may generally be applied in the animal kingdom to prolong survival under adverse conditions.Furthermore, this study proved that metabolic depression is tissue dependent. The general accepted view is that metabolic depression occurs in organs, such as muscle and alimentary tract, that are temporarily less important for the animal for survival. The question remains if vital organs, such as brain and heart, also express the mechanism of flexible metabolic depression under adverse environmental conditions. In the future we hope to elucidate this matter via 31P-NMR.
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ACKNOWLEDGEMENTS |
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Vincent van Ginneken was supported by a grant of the Life Sciences Foundation, which is subsidized by the Netherlands Organization for Scientific Research (NWO), SLW Project No. 427024. The in vivo NMR studies were carried out at the Netherlands in vivo NMR facility, which is at the Bijvoet Center of Utrecht University and is supported by the NWO.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: V. J. T. van Ginneken, Integrative Zoology, Dept. of Biology (EEW), van de Klaauw Laboratories, Kaiserstraat 63, 2311 GP Leiden, The Netherlands (E-mail: ginneken{at}rulsfb.leidenuniv.nl).
Received 17 April 1998; accepted in final form 7 July 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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