Download THE USE OF TBE ETHANOL PATHWAY IN GOLDFISH CARASSIUS

THE USE OF TBE ETHANOL PATHWAY IN GOLDFISH CARASSIUS AURA TUS (L.)
FOLLOWING ANOXIA
BY
I.P. ALELEYE-WOKOMA
Gatty Marine Laboratoty, Division of Environmental & Evolutional))
Biology, University of St, Andrews, Fife, Scotland
ABSTRACT
Goldfish Carassius auratus (L.) were subjected for a period of 6 weeks to 2h progressive
hypoxia followed by 6h anoxia in closed respirometers at 15oC. The concentrations of glucose,
lactate and ethanol were determined in whole gold fish C. auratus (L.) following exposure to both
hypoxia and anoxia.
Lactate accumulation (namol kg-I h-I) was 0.35 during the 1st week but declined to 0,14 in the 6th
week of exposure to ana,da. In contrast, ethanol excreted to the surrounding water, increased from
65% to 92% of the total production in the 1st and 6th week respectively. The switch fi-om lactate
accumulation to ethanol pathway utilization, with the resultant metabolic depression and anoxia
resistance is discussed,
Key Words: Anoxia tolerance, Ethanol excretion, Metabolic depression, Goldfish,
Carassius
auratus (L.)
INTRODUCTION
Gill breathers such as Trout, Tilapia, Perch and Haplochrornis are unable to survive habitats of low
oxygen. However, several African species of fish have accessory respiratory devices which enable
them to flourish in the dense swamps and muddy vegetation that are mainly anoxic. For emiple, the
lung of protopterus and polypterus and the diverticulate of the gill cavities in most the cat-fish
gennus Clarias make them air-breathers (Johannsen, 1970). By contrast, species such as rainbow
troyt and plaice are depeendent only on glycolysis for the production of ATP during hypoxias,
resultin in a lange lactate load (Dunn 8cHochachka, 11986: Wardle, 1978,Wokoma, 1990) and the
consezquent detrimental metabolic acido sus.
Three important observation have provide the basis for the study of the metabolism of
goldfish carassius auratus (L) durinng anoxia: (1) Lactate production was less than exxpected: (11)
ttue metabolic carbon dioxide was produce during anoxia;(111) the extraordinary ability of
golldfish to withsttannd prolonged periods of anoxia.
Crucian carp and goldfissh have shown a high tolerance to anoxia without produchig high
lactate concentration (vann den Thillatt 1977; Johnston and Bernard, 1983) Van den Thillart and
Kesbeke (1978) measured the production rates and wholee fish concentration of carbon dioxide,
ammonia, lactate, and glycogen, in goldfish held at compplete anoxia (3% air saturation) for 10
hours. The results showed that glycogen wasa oxidized to co2, suggesting that an unknown
electron acceptor must be involved in anaerobic energy production. Shoubridge et al; (19800,
discovered that the decreased lactate production from glycogen depletion,obsewed in anoxic
goldfish was due to firrther metabolism of pyruvate to ethanol. Unlike anaerobic glycolysis, tb.e
ethanol pathway does not result in accumulation of an acid end product, but instead, neutralethanol
is excreted to the surrounding waterwithout disturbingthe redox balanceof fish (Shubridge et al
1980; Mourik et al; 1982). theaim of the presentstudy was to determine whether periodic exposure
89
to anoxia could induce alcohol dehydrogenase and alter the balance between the proportions of
energy needs met by the lactate and ethanol pathways.
MATEIUALS AND METHOD
Goldfish (Carassius auratus L.), body weight 7.2 0.4 g, and standard length5.8 0.2 cm
(Mean S. E., n - 47) were obtained from commercial supplies. One group was maintained in
aerated waterat 15oC and the other, acclimated to periodic anoxia exposure.
The holding tank for fish consisted of a 10 litre tank of recirculated aerated water at 15oC.
Waste products were removed by circulating the water through a charcoal external filter containing
layers of coarse gravel, glass wool and activated charcoal.
Temperature was maintained by
pumping antifreeze glycol though a series of glass coils.
Opaque, open respirometer boxes (6x7x10cm) were used for the experiment Each box
had a perspex lid with two small holes, (one for delivery of nitrogen and the other for sampling the
water). Respirometer boxes were immersed in the holding tank to maintain a similar temperature
of 15oC.
Fish were transferred to the respirorneter boxes in groups of 4, 24 hours before the
measurement of o7gen consumption in order to reduce stress. Subsequenity, the respirometers
were sealed, and the respiration of the fish was used to reduce the oxygen tension (2 to 3h). Serial
water sarnples were collected for measurement of PW02 with Rank Brothers Oxygen Electode
(Bottisham, Cambridge, England). Nitrogen was then bubbled through (10 to 15 minutes) to
further reduce the PW02 to zero. One group of fish were held under an.oxic condition for a further
period of 6h and later returned to aerated water for recovery. The experiment was repeated every
48b.
Fish were stunned by a blow to the head, followed by spinal transection a,nd decapitation.
In a separate series of experiments samples of superficial red and deep white myotomal muscles
were rapidly excised from anaesthesized goldfish (6 fish, mean body wt. 7.0g± 0.4 and freezeclamped in brass tongs pre-cooled in liquid nitrogen (-196oC).
Glucose was analysed according to Benneyer and Bernt (1965). Lactate was measured by
the method of Hohorst (1965).
Ethanol was measured from the percbloric acid neutralized tissue extract or water sample.
The method is based on the reduction of pyridine nucleotide in a medium containing (mmol L-1),
nucleotide adenine clinucleotide, (NAD+), 1.8; Alcohol dehydrogenase, 150 units in glycine buffer
pH 9.0 at a wavelength of 340nun using a double beam spectroph-tometer.
Lactate Dehydrogenase (LDH) (E.G. LI, 27) was measured in the medium containing
(mmol L-1): Immidazole, 50. KCN, 2,5; NADH, 0.15: pyruvate, 10: pH 7.3 at a wavelength of
340nm.
Alcohol dehydrogenase (ADH) (E.0 1.1.1.1) was determined at 340nrn, in a medium
containing (mmol L-1) phosphate buffer, pH 7.0, 100; acetaldehyde, 3.6; NADH, 0.20; reduced
glut athione, 1.0.
Statistical Analysis
Data from fish acclimated to aerated and hypoxic water were compared using a one way
analysis of variance for unequal numbers.
Results
Routine oxygen consumption of 7.2g goldfish was 26.8±1.3m1. kg-1 h-1 at SiP This is
equivalent to an ATP production from aerobic metabolism of 8.9mmol. ATP kg-1 h-1 assuming a
P/O ratio of 3. On closing the respirometers, the fall in water P02 was followed by reduction in
V02 of goldfish. Ethanol concentration was below the level of detection in control samples.
Hypoxia a,nd anoxia resulted in significant increases in glucose, lactate and ethanol (Table 1)
Ethanol was freely diffusable across the gills and accumulated in the surrounding water (Table 1).
Consequently, 65% of the anoxia induced - ethanol was excreted during the 1 st week, and rose to
90
92% in the 6th week (Table 1).
The ATP yield from lactate and ethanol (tissue and excreted) assuming 0.016mmol ATP
MG-1 lactate (Bennett and Licht, 1972), represents requirements for anoxia acclimation. This AlP
turnover (mmol ATP kg-1. h-1) 1.78 amounted to 20% of that from aeroble pathways under
normoxic basal condition at 15oC.
Anoxia induced alcohol dehydrogenase activities (ADH) (umoles substrate utilized g. wet
wt-1 min-1), 31.7±2.75 in the red muscle was 3.5 times greater than that of white muscle fibres.
However, activities of lactate dehydrogenase (LDH) declined from 61.12±2.15 to
37.5±1.25 in whole goldfish, during the extended periods of anoxia.
DISCUSSION
Studies which demonstrate constant glucose turnover in a number of teleost species
(Cornish and Moon, 1985; Weber et al. 1986; Dunn & Hochachka, 1987; schultz et al 1992)
suggest that gluconeogenesis contributes significantly to the overall rate of glucose production.
In this experiment glucose production increased from 0.41 to 0.65 mmol kg-1 h-1.
Although amino acids are potential sources' of glucose in teleost fishes (Suarez and Mommsen,
1987), lactate is the preferential gluconeogenic precursor in teleost hepatocyte preparations (Moon
et al 1985). Resynthesis of glucose and glycogen (Cori-cycle) does not proceed in carp and
goldfish muscle, because of low activity of gluconeogenic enzymes, phosphoenolpyruvate
crboxylase and pyruvate carboxylase (Van Den Thillart and Smit, 1984). The major metabolic
rates and the blood glucose is oxidation to carbon dioxide or conversion to lactate.
The primary adaptive strategy supporting anaerobiosis in fish is profound depression of
metabolic rate, lowering ATP requirements to a level that can be supported over an extended
period by less efficient fermentative pathways of catabolism (Storey, 1985; 1987).
As a result of these modified pathways, the major anaerobic end products by goldfish, C.
auratus during exposure to periodic anoxia are lactate and ethanol. This confirms the results of
recent studies on goldfish (Carassius auratus L.) (Shoubridge and Hochachka, 1980; Van Den
Thlllart, 1981) and crucian carp (Carassius carassius L.), (Johnston and Bernard 1983).
In the present experiment, during the 1st week of exposure to anoxia, the net lactate
accumulation was 2.18mmol kg body wt-1, but declined to 34% in the 6th week. The rate of the
metabolically-produced lactate depends on the activity pattern of the fish (Wood and Perry, 1985).
In some teleosts, retention and utilization within the white muscle mass is the dominant means of
disposal For example, Tang and Boutillier (1991) found that protons produced in rainbow trout
during intense exercise were cleared by metabolic processes within the white muscle compactment.
Interestingly, lactate and proton movements in muscle appear to be coupled in a carriermediated transport process (Mason and Thomas, 1988). Bilinski and Jonas (1972) have also
demonstrated that gills, liver and muscle are all able to oxidise 14C-lactate at high rates in vitro.
In contrast to lactate, the net ethanol concentration increased significantly form 6.6 to
12.8mmol kg-1 (tissue + excreted). Previous authors have shown that ethanol is generated from
blood-borne lactate (Shoubridge and Hochachka, 1983) and possibly protein catabolism (Van Den
Thirllart et al 1985); hence the rapid depletion of glycogen is avoided.
However Shoubridge and Hochachka, (1980) have utilized carbon monoxide-poisoned
goldfish to demonstrate that a proportion of the 14C-lactate injected into anoxia goldfish could
be recovered in the ethanol fraction. This provided the evidence for the conversion of lactate
to ethanol.
The ethanol pathway is based on pyruvate dehydrogenase complex of goldfish
mitochondria which is able to decarboxylate pyruvate to acetaldehyde under anaerobic,
conditions (Mourik et al 1982). Acetaldehyde produced is subsequently detoxified to the
neutral anaerobic end-product, ethanol; this occurs in the cytoplasm by alcohol dehydrogenase
particularly in the skeletal muscle, (Van Den Thillart, 1982).
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This is consistent with the
present findings. Alcohol dehydrogenase activity in this study showed a 3.5 - fold increase in
the red muscle than the white muscle (Table 1). The utilization of the ethanol pathway allows
redox balance to be maintained, and enables glycolysis to proceed without the accumulation of
an acid end-product hence metabolic acidosis is avoided. Interestingly, it has been linked to
carbon dioxide excretion (Shoubridge and Hochachka, 1980), in a functional-coupling
process, with no demonstrable increase in ATP yield (Van Den Thirllart, 1982 Mourik et al
1982; Portner, 1987).
In contrast to the increase in the activities of ADH, lactate dehydrogenase (LDH)
declined, indicating a decreased reliance on anaerobic glycogenolysis to support anoxia in
aoldfish This is supported by the discovery of Rahman and Storey (1988), that anoxia
modified the glycolytic enzymes phosphofructokinase (PFR) and Pyruvate kinase (PK) of
goldfish, resulting in the reduction in activity.
The utilization of the ethanol pathway is apparently accompanied by metabolic rate
depression. The energy expenditure during anoxia (calculated from lactate and ethanol
concentrations) of 1.78mmol ATP, represents 20% of that of routine activity under aerobic
conditions at 15oC, Previous studies showed that C. auratus exposed to anoxia reduced their
metabolic rate to a level of 20 - 33% of the normoxic basal rate (Anderson, 1975; Van Den
Thirllart et al 1976; Shoubridge et al 1983). It seems that a reduction in metabolic rate is
essential in extending anoxic tolerance particularly at low temperatures. This is supported by
anoxia tolerance in goldfish, ranging from 16h at 20oC to several weeks ast 0oC (Van Den
Thirllart, 1977; Walker and Johansen, 1977). Similarly, crucian carp Carassius carassius (L.)
is also known to have survived the long winter months of frozen ponds that are considered
anoxic (Blazka 1958, Hyvarinen et al 1985; Piironen et al 1986), while all other species of fish
died.
In conclusion, this is the first experimental evidence that exposure to periodic anoxia
resulted in the induction of alcohol dehydrogenase activity which catalyzes the ethanol
pathway. The result is the excretion of 65 to 92% of the ethanol produced to the surrounding
water. The utilization of the ethanol pathway, although wasteful of carbon, prolongs the
survival of C. auratus beyond the range observed in other vertebrates.
Ackti owledgement
I am grateful to Prof. I.A. Johnson for guidance and Prof. S.N. Okiwelu for useful
com ents on the manuscript.
RE'FERENCES
Anderson, J. (1975):
Bennett, A.F. and Licht (1972):
Anaerobic resistance of Carassius auratus L. Ph.D thesis,
Australian National University.
Anaerobic metabolism during activity in lizards. J Comp.
Physiol., 81. 277-288.
Bergmeyer, H.Y. & Bernt, E. (1965): D. Glucose: determination with glucose oxidase and
peroxidase. in Methods of Enzymatic Analyses, (ed,
Bergmeyer) pp. 123-130 New York: Academic Press.
Bilinski, E., and Jonas, R.E.E. (1972): 0>ddation of lactate to Co2 by rainbow trout tissues. J
Fish Res. Bd. Can. 29: 1467-1471.
Blazka, P. (1958):
The anaerobic metabolism of fish. Physiol. Zool. 31, 117128.
Cornish I. and Moon, T.W. (1985): Glucose and lactate kinetics in American eels, Anguilla
rostrate. Am. J Physiol. 249, R67-R72.
Dun, J.F., and Hochachka, P.W. (1987): Turnover rates of glucose and lactate in rainbow trout
92
Hohorst, II.J. (1966):
during acute hypoxia. Can. J Zoo. 65, 1144-1148.
L (+) Lactate determination with lactate dehydrogenase and
DPN, In Methods of Enxymatic Analyses (ed. H.V
Bergmeyen) pp. 266-270. New York: Academic Press.
Hyvarinen, H., Holopainen, I.J. & J. Piironen. (1985):
Anaerobic wintering of crucian carp, (Carassius carassius
(L.) Atmual dynamics of glycogen reserves in nature
Comp. Biochern, Physiol. A. Comp. Physiol. 82 A: 797.Johansen, K. (1970):
803.
Air breathing in fishes in Fish Physiology Vol. 4 Hoar, W S,
and Randall, D.J. eds., Academic Press New York p. 361411.
Johnston, LA., and Bernard, L. (1983): Utilization of the ethanol pathway in carp following
exposure to anoxia. J. Exp. Biol 104, 73-78.
Mason, M.J., and Thomas, R.C. (1988): The microelectrode study of the mechanisms of Llactate entry into and release from frog satorius muscle. J
Physic). (Lond.) 400, 459-479.
Moon, T.VV.,Walsth, P.J., and Mommsen, T.P. (1985):
Fish hepatocytes: a model metabolic system. Can J. Fish.
Aquat. Sci. 42, 1772-1782.
Mourik, J., Raeven, P., Steur, K. & Addink, A.D.T. (1982):
Anaerobic metabolism of red skeletal muscle of goldfish,
Carassius auratus (L).
Mitochondrial produce
acetaldehyele as anaerobic electron accepton. FEBS
Letters 137, 111-114.
Piironen, J. & Holorainen, I.J. (1986): A note on the seasonality in anoxia tolerance of
crucian carp, (Carassius camssius L.) in the Laboratory.
Ann. Zool. Fenn. 23, 335-338.
Portner, H. (1987):
Contributions of anaerobic metabolism to pH regulation
in animal tissues: Theory J. Exp. Biol., 131: 69-87.
Rahtnan, M.S. & Storey, K.B. (1988). Role of Covalent
modification in the control of glycolytic enzmes in
response to environmental anoxia in goldfish.
J Comp. Physiol. 157, 813-820.
Schultz, P.M. Moyes, C.D., and Hochachka, P.W. (1992): Integrating metabolic pathways
in post-exercise recovery white muscle J Exp. Biol.
166' 181-196.
Shoubridge, E.A. & hochackha, P.W. (1980): Ethan.ol: Novel and product of verterate
anaerobic metabolism Sc. 209, 308-309.
Shoubridge, E.A. & Hochachka, P.W. (1983): The integration and control of metabolism
Storey, K.B. (1985):
in the anoxic goldfish Molee. Physiol. 4, 165-195,
A re-evaluation of the Pasteur effect; New mechanisms
in anaerobic metabolism. Molec. Physiol & 439-461.
Storey, K.B. (1987):
Tissue - specific controls on carbohydrate catabolism
during anoxia in goldfish. Physiol Zool. 60, 601-607.
93
Suarez, R.K., and MMosen, 'LP. (1987): Gluconeogenesis in teleost fishes Can. J. Zoo!.
65, 1869-1882.
Tane, Y and Boutilier, RIG. (1991): White muscle intracellular acid-bse and lactate status
following exhaustive exercise: a comparism between
freshwater and seawater adapted rainbow trout. 156, 153171 J Exp. Biol.
Van Den Thillart, G. (1977):
In: Influence of oxygen availability ori the energy
metabolism of goldfish, Carassius auratus (L) Ph.D thesis.
State University of Leidon, The Netherlands.
Van Den Thillart, G. (1982):
Adaptations of fish energy metabolism to hypoxia and
anoxia. Molec, Physiol. 2, 49-61.
Van Den Thillart, G. & De Bruin (1981):
Influence of environmental temperature on mitochondrial
membranes Biochim Biophys. Acta, 640, 43 9-447
Van Den ThiIlart, G. & Kesbeke, E. (1978): Anaerobic Production of carbon dioxide and
ammonia by goldfish Carassius auratus (L). Comp
Biochem. Physiol, 59A, 393.
Van Den Thillart, G. and H. Sinit. (1984): Carbohydrate metabolism of goldfish, Carassius
auratus (L). effects of long tema hypoxia acclimation on
enzyme patterns of red muscle, white muscle and liver J
Comp. Physiol. 154, 477-486.
Van Den Thillart, G., and Van Waarde, A. (1985): Teleosts in hypoxia; aspects of anaerobic
metabolism. Motec. Physiol. 7, 393-409.
Walker, R.M., and Johansen (1977): Anaerobic metabolism of goldfish Carassius auratus (L.)
Waldle, C.S. (1978):
Can. J. Zoo. 55, 304-311.
Non release lectic acid from anaerobic swimming muscle of
plaice, Pleuronectes platessa (L.) A stress reaction. J. Exp
Biol. 77, 141-155.
Webe J.M. Brill, R.W. and Hochaehlut, P.VV. (1986):
Mammalian metabolite flux rates in a teleost: Lactate atad
glucose turnover in a tuna. Am. J. Physiol. 250: R452Wolunna, I.P.A. (1990):
R458,
The effects of temperature and hypoxia on the metabolism
of Fishes. Ph.D Thesis. University of St. Andrews
94
Table 1:
Metabolite changes in whole goldfish (Carassius auratus L.)
following acclimation to either aerated water or progressive anoxia
exposure. Values represent (umol g-1) M±S.E.
1st Week
{PRTVATE }
Metabolite
Control
6th Week
2 h hypoxia
6 h anoxia
2h
6 h anoxia
hypoxia
Glucose
0.9±0.9
3.43±0.33
5.92±0.2
10.7±0.9
14.6±0.5
Lactate
0.83±0.11
0.93±0.05
3.10±0.21
0.34±0.09
1.09±0.04
Ethanol
0.76+0.03
3.06+0.28
0.08+0.03
1.04+0.15
(6h) Ethanol
excretion
4.3
(6h) Total
ethanol
(tissue and
excretion)
11.09
2.3+4.3=6.6
0.96+11.09=12.05
95