Anaerobic energy metabolism of the European eel,Anguilla anguilla L

Journal of Comparative Physiology. B

J Comp Physiol (1983) 149:469-475

9 Springer-Verlag ]983

Anaerobic Energy Metabolism of the European Eel, Anguilla anguilla L. Aren van Waarde*, Guido van den Thillart, and Fanja Kesbeke Department of Animal Physiology, Gorlaeus Laboratories, University of Leiden, P.O. Box 9502, NL-2300 RA Leiden, The Netherlands Accepted September 29, 1982

Summary. Eels, acclimated to 15 ~ and aerated water (Po2 130 mm Hg) were exposed to hypoxia (Po2 lowered from 130 to 8 mm Hg in 4 h) and to complete anoxia until loss of equilibrium. Experiments were carried out at night. The mean survival time (LTso) during anoxic conditions proved to be 5.7 h. ATP, ADP, AMP, IMP, CrP, glycogen, lactate, pyruvate, ~-ketoglutarate, malate, succinate, alanine, aspartate, glutamate and ammonia levels were determined in skeletal muscle and liver of control, hypoxic and anoxic fish. Some of the mentioned parameters were also measured in heart muscle and blood. Hypoxia causes declines of aspartate (muscle), CrP (muscle) and glycogen (liver, heart), and increases of alanine (blood, liver) and lactate (blood, liver, heart). During anoxia, muscle CrP stores are almost completely exhausted and adenylates are partially broken down to IMP. A decrease of glycogen and an accumulation of lactate were observed in all tissues examined. The energy charge of muscle and heart did not drop below 0.79, but in liver tissue it decreased from 0.65 to 0.17. Liver cytoplasm became significantly reduced during anoxia, but such a change of redox state did not occur in muscle. Eels seem to lack the capacity for anaerobic fermentation of glycogen to ethanol, as observed in goldfish. Lactate glycolysis and creatine phosphate breakdown appear to be the main energy producing pathways during anaerobiosis.

* Present address: Department of Cell Biology and Morphogenesis, Zoology Laboratory, University of Leiden, P.O. Box 9516, NL-2300 RA Leiden, The Netherlands Abbreviations: A L A alanine; A S P aspartate; CrP creatine phosphate; EC (adenylate) energy charge; GLU glutamate; GLC glucose; GL Y glycogen; I M P inosine-5'-monophosphate; ~KG ~ketoglutarate; L A C lactate; M A L malate; P Y R pyruvate; SUC succinate; T A N total pool of adenine nucleotides

Introduction

Metabolic and enzymatic studies on the influence of decreased oxygen availability on fish energy metabolism have been performed in a number of species, including crucian carp (Blazka 1958; Johnston 1975a), common carp (Driedzic and Hochachka 1975), rainbow trout (Burton and Spehar 1971; Johnston 1975b), bluegill sunfish (Burton and Spehar 1971; Heath and Pritchard 1965), flounder (Jorgensen and Mustafa 1980a, b) and goldfish (Walker and Johansen 1977; van den Thill a r t e t al. 1976, 1980, 1982; Mourik et al. 1982; van Waarde et al. 1982). The European eel, Anguilla anguilla L., however, has not yet been studied with respect to its anaerobic capabilities. The eel is an interesting object of study, since in its natural habitat, it is regularly exposed to hypoxic conditions, not only during winter when lakes, ponds and small streams are frozen, but probably also during summer because it feeds and buries itself in the mud. Therefore in the present paper we have initiated an investigation of the effects of hypoxia and anoxia on eel energy metabolism. The influence of decreased oxygen availability on tissue glycogen stores, phosphorylated compounds, Krebs-cycle intermediates, anaerobic end products and some free amino acids are described and compared to results obtained for other species of fish. Skeletal muscle, heart muscle, liver and blood were chosen as objects of study because of their importance in energy metabolism. Normoxiaacclimated fish were exposed to either hypoxic or anoxic conditions. In the latter experiments, a period of hypoxia was followed by several hours of complete anoxia. Materials and Methods Animals. Experiments were carried out in autumn 1981 with

healthy eels (mean weight 80 g) which had been caught in the

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A. van Waarde et al. : Anaerobic Energy Metabolism of the European Eel

' K a a g ' lakes near the city of Leiden by local fishermen. The animals were acclimated to 15 ~ normal oxygen levels (Po~ = 130 m m Hg) and a 16 h light period. As in the laboratory the eels did not take up food, the animals were used after a relatively short acclimation period of three weeks.

Conditioning and Experimentation. Experiments were performed during the dark period at a temperature of 15 ~ in a respirometer as described by van den Thillart and Kesbeke (1978) containing 5-15 fish. After a 24 h period of habituation to the apparatus, three experimental conditions were administered: control (Po2 kept stable at 130 mm Hg), hypoxia (Po~lowered from 130 to 8 mm Hg in 4 h) and anoxia (as the former, but Po~ lowered to zero; animals were removed from the respirometer at the moment of loss of equilibrium). Experiments were terminated by injection of a solution of the anaesthetic MS 222 into the respirometer (final concentration 100 ppm). In preliminary experiments, we found eels to be rather resistant to MS 222, since in the presence of anaesthetic the animals cease to ventilate. Therefore eels were taken by hand out of the respirometer 15 rain after MS 222 administration and their gills were flushed with an aerated or anoxic 100 ppm MS 222solution (the oxygen tension depending on the type of experiment). When anaesthesia had become complete, the body wall was opened and a blood sample was taken by cardiac puncture. Before removal of muscle, animals were intracardially injected with a solution of curare (2 mg/kg body weight in 1% NaC1). After 10 min (allowing the curare to be pumped through the whole body) a sample of muscle was very rapidly removed and freeze-clamped as described by van den Thillart et al. (1982). After removal of the muscle, heart and liver were processed in the same way. The extraction of deep-frozen tissues was performed as described by van den Thillart et at. (1982), but samples for glycogen determination were extracted with 9 volumes of ice-cold 6% HC104.

Determination of Intermediates. Levels of intermediates were determined in neutralized perchlorate extracts by means of spectrophotometric methods. The following assays were used: IMP was determined as described by van den Thillart et al. (1980), ammonia by the method of Da Fonseca-Wollheim (1973), pyruvate and ~-ketoglutarate according to Williamson et al. (1967), lactate by the method of Gercken (1960), succinate by the method of Michal et al. (1976), glycogen according to Fraser et al. (1966), creatine phosphate, ATP, ADP, AMP, glutamate, malate, glucose and aspartate by methods described in Bergmeyer (1970). Ethanol was determined with a commercial test kit available from Boehringer Mannheim GmbH, FRG.

Determination of Enzyme Activities. Alcohol dehydrogenase activity was assayed as described by Mourik et al. (1982).

Results

Survival Experiments A s s h o w n in Fig. 1, the L T s 0 f o r c o m p l e t e a n o x i a at 15 ~ p r o v e d to be 5.7 h. A n i m a l s w e r e r e m o v e d f r o m the r e s p i r o m e t e r a n d c o n s i d e r e d ' d e a d ' w h e n t h e y h a d lost e q u i l i b r i u m , b u t a c t u a l l y m o s t fish r e c o v e r e d w h e n t h e y were t r a n s f e r r e d to o x y g e n a t e d water.

Anoxio-toLeronce ofeuropeoneel 15- e o tl

,c 10 o

0

I

~

5

110

I

,

Time(h)

Fig. 1. Survival of eels during exposure to anoxia. Freshwater

eels, weighing about 80 g and acclimated to 15 ~ were transferred to a respirometer and kept at 15 ~ and a Po2 of 130 Torr for 24 h. Environmental oxygen was then depleted by oxygen consumption of the fish after closing of the water inlet. Anoxia was reached in about 4 h. The onset of anoxia was determined by extrapolation of the Po2 decline from 40 to 20 Torr. The fish were examined every 30 min and taken out when completely relaxed. Twelve fish (nos, 3-14) were used for metabolite measurements

Tissue-Specific Changes of Intermediates Skeletal Muscle. I n the e x p e r i m e n t a l eels, we c o u l d n o t r e c o g n i z e s e p a r a t e z o n e s o f red a n d white m u s cle fibers, as f o r e x a m p l e in c a r p , g o l d f i s h o r r a i n b o w trout. T h e r e f o r e , an epaxial piece o f m u s cle (size a b o u t 20 x 8 x 7 r a m ) w a s r e m o v e d j u s t b e h i n d the p e c t o r a l fins in all s p e c i m e n s e x a m i n e d . T h e laterial line r e g i o n w a s a v o i d e d . As s h o w n in T a b l e 1, m o s t p a r a m e t e r s are n o t c h a n g e d b y a s h o r t p e r i o d o f severe h y p o x i a . W e only observed a 50% reduction of CrP, 60% reduction of ammonia and a 75% reduction of aspartate. Long-term anoxia induces more profound c h a n g e s in t h e levels o f m u s c l e m e t a b o l i t e s . A t the m o m e n t o f loss o f e q u i l i b r i u m , C r P stores are exh a u s t e d f o r m o r e t h a n 9 2 % a n d the a d e n y l a t e ene r g y c h a r g e has d r o p p e d b e l o w 0.8 d u e to a r e d u c tion of ATP-levels and an accumulation of AMP. T h e (statistically n o n - s i g n i f i c a n t ) b r e a k d o w n o f the a d e n y l a t e p o o l is a c c o m p a n i e d by a significant production of IMP. Concentrations of ammonia h a v e r e t u r n e d to the c o n t r o l level, while a s p a r t a t e c o n t e n t r e m a i n s low. A l t h o u g h the level o f lactate is significantly, i n c r e a s e d f r o m 2 to 9 g m o l e s / g , l a c t a t e / p y r u v a t e ratios are u n c h a n g e d d u e to a n e q u i v a l e n t i n c r e a s e o f p y r u v a t e , i n d i c a t i n g a n unc h a n g e d c y t o p l a s m i c r e d o x state d u r i n g a n o x i a . G l y c o g e n stores are d e p l e t e d f o r a b o u t 7 0 % . Levels o f g l u t a m a t e , m a l a t e a n d a l a n i n e are n o t in-

A. van Waarde et al. : Anaerobic Energy Metabolism of the European Eel

471

Table 1. Influence of hypoxia and anoxia on the concentrations of intermediates in eel tissues. Levels are expressed as nmoles/g wet weight. The energy charge (EC) value has no dimension. Mean values -+S.D. @=4). Differences between groups were tested by Wilcoxon's Q-test. T A N Total level of adenine nucleotides. Glycogen levels are expressed as nmoles glucose units/g wet weight Control

Hypoxia

Anoxia -

Muscle

CrP ATP ADP AMP TAN EC IMP NH 3 PYR ~KG GLY LAC MAL SUC ALA ASP GLU LAC/PYR GLU/ ~KG.NH +

Hypo•

Anoxia

Liver

15,410+-4,900 1,790_+ 190 330_+ 70 50+- 20 2,160__. 240 0.90_+ 0 . 0 1 120+ 160 910-+ 50 60+- 50 17-+ 4 1,800_+ 720 2,390_+ 460 130+- 50 690+- 470 1,030-+ 420 140_+ 90 510_+ 120 76_+ 33+

76 10

7,920+_2,260* 1,560_+ 360 330_+ 80 50_+ 20 1,930_+ 410 0.89_+ 0 . 0 3 50_+ 40 350+ 80" 100_+ 40 40-+ 26 1,420_+ 690 3,280_+1,270 120-+ 30 1,040_+ 740 820-+ 300 35- 13" 510_+ 110 34_+ 58_+

8 47

1,210+-1,020" 1,140_+ 500 340_+ 50 160_+ 50* 1,630_+ 530 0.79_+ 0.09" 620+ 300* 1,110_+ 320 210_+ 130" 36_+ 21 530-+ 150" 8,780_+2,130" 120_+ 20 1,680-+ 590 840-+ 290 25+ 10* 480_+ 150 61_+ 15-+

45 7*

Heart

ATP ADP AMP TAN EC IMP GLY LAC

Control

ATP ADP AMP TAN EC IMP NH 3 PYR o~KG GLY LAC MAL SUC ALA

950-+ 260 620_+ 170 630_+ 80 500+. 190 370_+ 220 500_+ 180 1,950_+ 230 1,620_+ 260 0.65_+ 0 . 1 1 0.54+ 0 . 1 1 160-+ 210 50-+ 40 4,280_+ 620 1,940-+ 270* 27_+ 12 81 • 14" 55_+ 11 63_+ 36 14,510_+13,330 6,110_+5,550 290_+ 160 1,760_+ 940* 770_+ 290 1,120_+ 390 4,800_+3,890 10,340_+3,240 430_+ 160 1,860_+ 360*

110___ 120" 220_+ 90* 870+- 260* 1,200_+ 390* 0.17_+ 0.09* 140_+ 20 2,030_+ I90" 50_+ 40 73_+ 49 1,230_+ 10" 3,660_+1,490' 760_+ 330 9,910-+3,760 2,390_+ 490*

GLU

3,040_+1,540 3,t90_+2,190

2,320_+1,610

LAC/PYR GLU/ ~xKG.NH +

10_+ 14_+

3 9

23_+ 35_+

14 39

108_+ 18•

60* 11

Blood

850 (n=2) 230 (n=2) 60 (n = 2) 1,140 (n=2) 0.85 (n=2) 260 (n=2) 4,940-+3,020 1,090 (n=2)

820 (n=2) 220 (n=2) 20 (n = 2) 1,050 (n=2) 0.87 (n=2) 150 (n=2) 2,030 (n=2) 2,800 (n=2)

440_+ 370 110_+ 80 50 i 30 600_+ 470 0.79+ 0.06 250_+ 100 360_+ 60* 4,830_+2,530

NH3 LAC ALA GLU GLC

770-+ 320

450-t- 130

400-+ 200

770-+ 530 230+ 80 310_+ 250

3,910-+1,520" 640-t- t70" 310-+ 170

7,720_+1,330" 440_+ 140" 420-+ 180

3,720_+ 380

2,230+1,030"

6,380_+2,920*

* Significant difference at the 5% level with respect to the control animals

Table 2. Concentration gradients of anaerobic end-products Parameter

Control

Hypoxia

Anoxia

+0.14_+0.33

--0.10 _0.09

+0.71_+0.14"

+1.62_+0.93

-0.63_+1.13"

+1.06_+1.08

+3.51_+0.59

+1.50_+0.32"

1.68_+0.30"

-0.49_+0.47

-2.15_+0.82"

-4.09_+0.89*

Muscle

Ammonia gradient Lactate gradient Liver

Ammonia gradient Lactate gradient

Concentration gradients are expressed as gmoles/g. They were calculated as the difference in concentration betweerl tissue and blood. Results are presented as the means_+ standard deviations of 4 independent observations. Differences with respect to the control ~ o u p were tested by Wilcoxon's Q-test; significant differences (at the 5% level) are indicated by an asterisk (*)

fluenced by the oxygen availability. Succinate levels tend to increase, although this increase is not statistically significant. The value for the redox couple of the glutamate dehydrogenase-catalyzed reaction is significantly reduced, indicating a more oxidized state of the mitochondrial matrix during anoxia. Ammonia gradients over the muscular cell membrane are presented in Table 2. In the control and hypoxic groups these are very low, but during anoxia they show a fivefold increase. Lactate gradients are positive in control and anoxic groups, but negative during hypoxia, probably because during hypoxic conditions anaerobic glycolysis is activated in muscle less rapidly than in other tissues. Liver. Liver tissue appears to be more sensitive to hypoxic stress than skeletal muscle. Ammonia

472

A. van Waarde et al. : Anaerobic Energy Metabolism of the European Eel

concentration is decreased to less than half of the control and stays at that level during the subsequent anoxic period. The levels of pyruvate, lactate, succinate and alanine are significantly increased. Glycogen is depleted by about 60%, however, standard errors of the mean in the control group are extremely large, making comparisons difficult. If exposure to anoxia becomes lethal (mean duration 5.7 h) dramatic changes occur in the levels of several intermediates. ATP and ADP are broken down to 12 and 35% of the control values, while AMP shows a more than twofold increase. As a consequence, there is a severe drop of the energy charge value from 0.65 to 0.17. The significant decline of the adenylate pool is not accompanied by any increase of IMP. Lactate and alanine levels show a further increase, but the succinate level is not further increased in comparison to the hypoxic group. Levels of glutamate, malate and ~-ketoglutarate are not influenced by oxygen availability. Glycogen stores drop to 9% of the control value. Lactate/pyruvate ratios are significantly increased during anoxia, indicating a reduction of the redox state of liver cytoplasm. In contrast, the redox state of the glutamate dehydrogenase-catalyzed reaction is not changed by anoxia, which implies a rather stable redox state of the mitochondrial matrix. The ammonia gradient over the liver cell membrane is significantly decreased by hypoxia and shows no further change during anoxia. Lactate gradients over the cell membrane are increased due to hypoxia and become even more negative during anoxia.

Heart Muscle. The only changes observed in heart muscle during hypoxia are an almost threefold increase of lactate and a 60% reduction of glycogen content. During anoxia, both the ATP- and ADPlevels are reduced to half of the control value, while AMP does not accumulate. The energy charge does not drop below 0.79 and the muscle retains its ability of contraction even when equilibrium has been lost after 5.7 h of anoxia. The almost 50% reduction of the adenylate pool is not accompanied by any increase of IMP. Although lactate levels show a fivefold increase during anoxia, lactate gradients over the cell membrane remain negative. Blood. Blood ammonia levels tend to be lower during anoxia, however, this tendency is not statistically significant. Lactate levels show a fivefold increase during hypoxia and are raised tenfold above the control value during the subsequent 5,7 h period of anoxia. Hypoxia causes a 2 to 3-fold increase

of alanine levels, while those of glutamate remain unchanged. Blood glucose shows a transient decrease during hypoxia, but its level rises twofold above the control during the subsequent period of anoxia, probably because of mobilization of liver glycogen. Discussion

Directly Available Energy ATP-levels in muscle and heart are not significantly altered by hypoxia. In muscle, the ATP content is probably kept stable by rapid transphosphorylation of ADP at the expense of CrP, since CrP stores are depleted by almost 50%. The strong adenylate breakdown in the liver is probably related to the absence of large CrP stores in this tissue. In all tissues examined, anoxia causes a decline of the directly available energy stores (sum of ATP and CrP), indicating that energy demands exceed the capacity of energy production via anaerobic processes.

Adenylate Energy Charge As shown in Table 1, the energy charge values of heart and skeletal muscle are not changed by a short period of severe hypoxia, probably because of the buffering effect of creatine phosphate. During anoxia, however, the adenylate energy charge is significantly lowered in all tissues examined. In skeletal muscle and heart, energy charge values do not drop below 0.79. This stabilization appears to be brought about by the simultaneous action of at least 3 mechanisms: a) rephosphorylation of ADP via creatine kinase, resulting in depletion of the CrP store; b) rephosphorYlation of ADP by anaerobic glycolysis, leading to glycogen breakdown and accumulation of lactate; c) breakdown of AMP, as shown by a decline of the size of the adenylate pool. In skeletal muscle, this process seems to be catalyzed by adenylate deaminase, since it gives rise to accumulation of IMP. In heart muscle, it may either be catalyzed by 5'-nucleotidase (leading to the accumulation of adenosine, which may partially leak out into the blood), or by AMP-deaminase, 5'-nucleotidase and nucleoside phosphorylase (leading to accumulation of inosine and hypoxanthine), since in this tissue IMP-accumulation cannot be detected. Accumulation of inosine and hypoxanthine is a process known to occur in the postmortem degradation of nucleotides in many fish species (Spinelli 1967; Kassemsarn et al. 1963).

A. van Waarde et al. : AnaerobicEnergyMetabolismof the European Eel The energy charge in the liver is very sensitive to hypoxia and anoxia. Anoxia causes a decrease from 0.65 to 0.17. Similar tissue differences with respect to the response of energy charge to decreased oxygen levels have also been described for goldfish by van den Thiltart et al. (1980) and for flounder by Jorgensen and Mustafa (1980b).

Glycogen Depletion and Accumulation ofEnd-Products Anoxia causes a depletion of glycogen stores in all tissues examined, which is accompanied by accumulation of lactate (Table 1). Glycogen levels of eel tissues appear to be very low in comparison to those of the flounder (J~rgensen and Mustafa 1980a) or goldfish (van den Thillart et al. 1980; Walker and Johansen 1977). This low glycogen level is probably not due to starvation, since at 8 ~ eels can be starved for at least 95 days without any decline of tissue glycogen content (Larsson and Lewander 1973; Dave et al. 1975). Levels of glycogen as observed in the present study are also very low in comparison to those reported previously in eels (Larsson and Lewander 1973 ; Dave et al. 1975). The reason for this discrepancy is not clear; however, eels used by the Swedish authors were caught and kept in seawater at 8 ~ while those of the present study came from freshwater and were kept at 15 ~ Because of this difference in conditioning, comparison is difficult. Our experiments point to the great importance of glycogen as an anaerobic fuel, since glycogen stores are depleted about 93% in heart muscle, 92% in liver and 70% in skeletal muscle (Table 1). Although changes in succinate concentrations with decreased oxygen availability are not statistically significant due to the high variances, succinate may be an important end-product. Succinate concentrations as observed in eel liver during anoxia are about 13-fold higher than those observed in goldfish under the same conditions (van Waarde et al. 1982). During hypoxia, succinate accumulation in eel liver is much greater than that of lactate. Although glycolysis is activated during anoxia, alanine levels of eel muscle do not show any increase, in contrast to data obtained previously in goldfish (van Waarde et al. 1982).

Redox Slate At tow Po2, the redox state of eel muscle does not shift to a more reduced level. Although the total pools of pyruvate and lactate increase in size, the lactate/pyruvate ratio is not changed, indicat-

473

ing stability of the cytoplasmic redox state. The mitochondrial compartment even appears to become more oxidized, as indicated by a decreased value of the redox couple of glutamate dehydrogenase (Table 1). In liver tissue, however, the cytoplasmic redox state (as indicated by the LAC/PYR ratio) becomes more reduced during anoxia. A change is not observed, though, in the mitochondrial compartment, since the value of the glutamate dehydrogenase redox couple appears to be independent of oxygen availability. A similar relationship between the redox state and oxygen availability has been observed in rat liver by Brosnan et al. (1970) and Sylvia et al. (1975). In goldfish, a stable redox state of muscle tissue during anoxia could be correlated with the capacity of this species to produce ethanol, acetaldehyde functioning as a hydrogen sink (Mourik et al. 1982; van den Thillart et al. 1982). In higher vertebrates, stimulation of anaerobic lactate production results in a 20-fold increase of the LAC/PYR ratio (see the discussion of van den Thillart et al. 1982). It should therefore be considered exceptional that eel muscle is able to stabilize the cytoplasmic redox state during anoxia.

Concentration Gradients of Anaerobic End-Products As shown in Table 2, ammonia gradients over the muscular cell membrane are close to zero in control and hypoxic animals, which suggests a small flux. During anoxia, ammonia gradients show a strong increase. The increase of ammonia concentration in muscle tissue during the transition from hypoxia to 6 h of anoxia is about equal to the increase of IMP. As a means to stabilize the energy charge, muscle AMP-deaminase converts AMP into IMP plus NH 3 (van den Thillart et al. 1980). The increase of ammonia level might be due to this mechanism when no ammonia was lost to the circulation, which is possible since white muscle is poorly perfused. In contrast to muscle, ammonia gradients over the liver-blood barrier are significantly decreased during hypoxia, which suggests a diminished ammonia production and efflux. A certain positive gradient, however, is maintained, which indicates that ammoniogenesis is not completely blocked. Since eel liver becomes rather reduced during anoxia, oxidative deamination could be inhibited, but ammonia might be produced by breakdown of the adenylate pool and via the asparaginase and glutaminase reactions.

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A. van Waarde et al. : Anaerobic Energy Metabolism of the European Eel

In goldfish, anaerobiosis is characterized by a negative gradient of lactate over the muscle cell membrane, which suggests a continuous influx of lactate from the blood. Although there is a very substantial glycogen breakdown (van den Thillart et al. 1980), lactate accumulation is rather moderate and is not stoichiometric with glycogen depletion (van den Thillart and Kesbeke 1978; van den Thillart et al. 1980). A solution for this problem has been presented by Shoubridge and Hochachka (1980), who discovered ethanol as an anaerobic end-product, which is excreted into the surrounding water, but only when the fish are kept anoxic (van den Thillart and De Wilde, unpublished). Goldfish muscle tissue contains a very active alcohol dehydrogenase with special kinetics, making it suitable for ethanol production (Mourik et al. 1982). Under anaerobic conditions, muscle pyruvate is decarboxylated in mitochondria by the pyruvate dehydrogenase complex. Acetaldehyde thus formed is reduced in the cytoplasm by alcohol dehydrogenase (Mourik et al./982). As suggested by Shoubridge (1982) on the basis of tracer experiments, lactate produced in other tissues may be transported to the goldfish myotome, where it is converted to ethanol. Ethanol diffuses out of the body and glycolysis may proceed to a very large extent without acidosis. The negative lactate gradient of goldfish muscle during anoxia, as observed by van den Thillart et al. (1982) is probably related to continuous removal of intracellular lactate by the ethanol-producing system. In eel muscle, however, lactate gradients during anoxia are positive (Table 2), which is inconsistent with influx from the blood. In eel muscle homogehates, alcohol dehydrogenase activity cannot be demonstrated (activity 0.001 gmoles.min- 1.g- 1 at 20 ~ Although eel liver shows low enzyme activity (0.3 gmol.min-1.g-1 at 20 ~ measured in the direction of ethanol oxidation), ethanol cannot be found in the surrounding water even after lethal duration of anoxia. Therefore, the eel does not seem to possess an ethanol-producing system as described for goldfish, and lactate seems to be the major end-product of anaerobic glycogen catabolism. Acknowledgements. We wish to thank Prof. Dr. A.D.F. Addink and Dr. H. Smit for their critical reading of the manuscript.

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