Physiology of Finfish and Shellfish (2+1)

4.4.5. Anaerobic metabolism

Animals that tolerate prolonged exposure to zero oxygen, must of necessity obtain their metabolic energy from non-oxidative reactions. This holds true for many intestinal parasites, for animals that live in the oxygen-free mud of lakes and pods, for bivalves that remain closed during long periods, and for many others.

Animals that can intermittently survive long periods of anoxic conditions are called facultative anaerobic organisms. An example is the sea anemone Bunodosoma, which is found along the Texas Gulf Coast. These sea anemones are often buried in the sand, and some are found under neath rocks where the sand is black due to anaerobic sulfur bacteria. The survival of these sea anemones in the absence of oxygen is impressive. Animals that were placed individually in sealed chambers containing sea water that had been flushed with nitrogen were left undisturbed in sealsed jars for 6 weeks. When the jars were opened, a pungent odour of sulfur was released, and there was no measurable oxygen left in the water. When the sea anemones were placed in aerated water, they all opened quickly and showed normal activity. Their neuromuscular responses were the same and showed the same threshold for stimulation whether or not oxygen was present (Mangum 1980).

How does an animal obtain energy to remain alive in the total absence of oxygen? One common process for obtaining energy under anaerobic conditions is the breakdown of carbohydrate into lactic acid. For example, 1 mol glucose can anaerobically be broken down to 2 mol lactic acid (C₆H₁₂O₆ -----> 2 C₃H₆O₃).

This process, known as glycolysis, occurs commonly in vertebrate muscle, when the energy demand in heavy exercise exceeds the available oxygen.

It will be convenient to discuss the needs for energy by using the common currency of energy metabolism, adenosine triphosphate (ATP). ATP is the immediate source of energy for nearly every energy-requiring process in the living organism. If we consider oxidative and anoxic metabolism from this viewpoint, the situation looks as follows.

When 1 mol glucose, through intermediate biochemical reactions, is degraded to 2 mol lactic acid, 2 mol ATP is synthesized and made available to supply energy. In contrast, the complete oxidation of 1 mol glucose to carbon dioxide and water yields 36 mol ATP (6 mol in the formation of 2 mol pyruvic acid and 30 mol in the complete oxidation of the pyruvic acid via the Krebs cycle). The yield from the anaerobic breakdown of glucose is therefore small, not quite 6% of what would be available from the complete oxidation of glucose.

Most anaerobic glycolysis depends on glycogen, rather than glucose, as the substrate. Glycogen is a polymer of glucose, and the single glucose unit in the polymer is called a glycosyl unit. If glycogen is the substrate in anaerobic metabolism, the yield is 3 mol ATP from the formation of 2 mol lactic acid from 1 mol glycosyl. The yield from anaerobic glycolysis, using glycogen as the substrate, therefore yields about 8% of what would be available from the complete oxidation of the substrate to carbon dioxide and water. The reason a glycosyl unit yields more energy than glucose is that some energy was used in the synthesis of the glycogen polymer from glucose, and this eliminates one energy-requiring step in the breakdown to lactic acid.

It is uneconomical to use anaerobic glycolysis when the yield of ATP is so low, but an intestinal parasite probably doesn’t care much about economy. However, animals that use glycolysis only intermittently can be expected to use lactic acid as a substrate for further oxidation (in the Krebs cycle) and thus in the end, gain the full energy value of the original carbohydrate substrate.

The tolerance to anoxic conditions sometimes is amazing. It has been reported that the crucian carp (Carassius carassius) can live for 5.5 months under the ice of a frozen lake when the water under the ice is oxygen-free because of fermentation of dead plant material and hydrogen sulfide is present (Blazka 1958). (Hydrogen sulfide is poisonous, because it binds to and inactivates cytochrome oxidase, but because no oxygen is present to permit oxidative metabolism, this does not poison the fish). These carp produce practically, no lactic acid in the tissues, and other metabolic processes. It was suggested that, in these carp the formation of fatty acids is the end result of metabolism, represented as an increase in the total amount of fat in the fish during the long period of anaerobic conditions.

The anaerobic metabolism of fish has an interesting consequence, that is, related to the low carbon dioxide concentration in fish blood. The low carbon dioxide concentration means that, the amount of sodium bicarbonate that serves as the main buffering substance in the blood is also very low. The addition of large amounts of lactic acid would therefore severely disturb the normal acid-base balance of the poorly buffered blood.

A trout that was made to swim vigorously for 15 minutes showed a considerable increase in the lactic acid concentration in the blood, but after it was allowed to rest the lactic acid surged to much higher levels (Black et al., 1966). Apparently, the extremely high demands on the swimming muscles were not met through oxidative metabolism, and lactic acid was formed. However, all lactic acid was not immediately released into the circulation but accumulated in the muscle, and only after the fish was allowed to rest and recover, the lactic acid is released. It could be then used by other organs for oxidative metabolism and for resynthesis of glucose and glycogen, a process that normally takes place in the liver.

Measurements of the buffering capacity of the muscle itself have given some interesting results. Muscles from a variety of fish and marine mammals showed that the highest buffering capacities were found in muscles capable of either intense bursts of glycolytic function or prolonged, low-level anaerobic activity. Among the fish studied, warm-bodied speices such as tuna had the greatest buffering capacities. Deep-sea fishes and shallow-living fishes with sluggish swimming habits and low buffering capacities. Marine mammals, on the other hand, had high muscle buffering capacities, and as we shall see later, diving mammals depend to a great extend on anaerobic lactic acid formation for the supply of energy to the muscles during underwater swimming (Castellini and Somero 1981).

We have so far discussed only glycolysis and formation of lactic acid in anaerobic metabolism; other reactions are available. Several different pathways have been established for a variety of organisms, and especially those invertebrates that can tolerate long periods of anoxia utilize a number of interesting processes (Hochachks 1980).

A study of goldfish, a close relative of carp, has given us further understanding of anaerobic metabolism in fish. Goldfish tolerate anoxia very well and can survive for several days in the absence of oxygen, especially undo low temperature condition. The amount of glycolysis, as indicated by a increase in lactic acid in muscle and blood, to support the metabolic rate is insufficient. Furthermore, even if there is a complete lack of oxygen, some metabolic carbon dioxide is formed (Shoubridge and Hochachka 1980).

To study these problems, goldfish were first poisoned with carbon monoxide to block the oxygen transport in blood and to inhibit cytochrome oxidase so that any oxygen remaining in the body (such as in the swimbladder) would be unavailable for oxidative metabolism. Later, the fish were moved to oxygen-free water and kept under nitrogen for 12 hours. After 12 hours the results were as shown in Table 6.2. Lactate had accumulated in the fish, but there was also a similar concentration of ethanol (ordinary ethyl alcohol) in the tissues. Furthermore, a substantial amount of ethanol was found in the water in which the fish were kept.

Several further observations indicated that the ethanol had been formed from lactate. After the first 12 hours the lactate concentrations in the fish increased only slowly, but no locate was found in the water, and if additional lactate had been formed, it apparently had been metabolized to ethanol. To confirm this suggestion, lactate labeled with radioactive carbon (¹⁴C) was injected, and 88 to 92% of the injected amount was collected as ethanol.

Is there any advantage to the formation of ethanol? The ethanol is easily removed from the body because it readily diffuses out through the gills. This is wasteful because ethanol has a considerable fuel value, but on the other hand it is important for fish that the lactic acid concentration should not reach too high levels. As we saw before, fish blood has a poor bicarbonate buffer system, which in turn is a consequence of the high solubility of CO2 in water. Transformation of lactate to ethanol, which is readily lost to the water, apparently serves as a means of avoiding acidosis, although the fish pays for this privilege in the form of excessive loss of metabolic fuel.

Many will wish to compare the ethanol concentration found in the goldfish with concentrations commonly found in humans after alcohol consumption. A human who has a blood alcohol level of 0.25% ethanol is intoxicated, and in many countries (but not all) the legal limit for automobile driving is one-fifth of this, or 0.05%. Any value lower than this should then not constitute being under the influence of alcohol; the concentration in the goldfish described in Table 6.2 was 0.024%, or one-half the legal limit.

Table 6.2 Concentration of lactate and ethanol in goldfish after 12 hours anoxia at 4^oC. (Shoubridge and Hochachka 1980)

Metabolite concentration (mmol per kg fish)
	Tissue lactate	Tissue ethanol	Ethanol in water
Control	0.18	0	0
Anoxia	5.81	4,58	6.63