Blood Gas Transport in the Cephalopod, Sepia Officinalis

The high aerobic metabolism of most cephalopods exceeds that for other aquatic invertebrates of similar size and also surpasses that of many fast-swimming fishes (Altman & Ditmer, 1971).

Since cephalopod blood has a low O₂ capacity, typically not exceeding 5 vol%, the high tissue O₂ requirements must be satisfied by high perfusion rates (cardiac outputs) and by maximum utilization of the blood-borne, circulating O₂ pool. The utilization of O₂ transported by the blood and the physiological significance of blood respiratory properties studied in vitro can never be fully assessed unless information about in vivo circulating levels of blood gases and pH are available from arterial and venous blood. Such information is extremely rare for cephalopods, due to the obvious technical difficulties associated with implanting chronically indwelling intravascular catheters in these animals. Studying the active squid, Loligo, Redfield & Goodkind (1929) measured near-maximal arteriovenous O₂ content differences by direct blood sampling from pinned-down, cut open and artificially ventilated specimens. Based on these measurements blood O₂ utilization in Loligo exceeded 90%. The authors implied that values of blood pH measured concurrently established that nearly 40% of the large O₂ turnover to the tissues depended on the uniquely high Bohr shifts in Loligo. The Bohr factor ϕ (Δ log P₅₀/ΔpH) was reported to be −1·8 fori Loligo (Redfield, Coolidge & Hurd, 1926).

Studies of the large Octopus dofleini at rest, based on blood samplings from chronically cannulated animals, showed O₂ utilizations averaging 72 % with a maximum of 85 % (Johansen & Lenfant, 1966). O. dofleini also has a large Bohr factor(ϕ = − 0·80) (Lenfant & Johansen, 1965).

Blood O₂ transport in the phylogenetically primitive Nautilus pompilius showed a much lower value for O₂ utilization of 35%. Nautilus is sluggish and has a small Bohr factor(ϕ = −0·20) (Johansen, Redmond & Bourne, 1978).

The study to be reported concerns an evaluation of blood gas transport in the common North-Atlantic cuttlefish, Sepia officinalis.

Seven specimens of Sepia officinalis, ranging in weight from 1500 to 1600 g, were used in the study. The animals were obtained by trawling in the English Channel near Plymouth. Before experiments, the animals had been kept for several days in well aerated running sea water at 17 °C. They were fed live crabs at intervals, but food was withheld for at least 24 h prior to anaesthesia.

Anaesthesia was induced by slow addition of ethyl alcohol to an aerated seawater bath to a final concentration of about 3 % in the sea water. Muscle relaxation occurred after about 30 min exposure to the alcohol-water mixture. The surgical procedures lasted about 20 min. The large vena cava cephalica, cannulated for mixed venous blood, is accessible slightly posterior to the anus when the mantle musculature is relaxed. A PE 60 polythene catheter was passed downstream through a hole made in the vessel wall with a cutting-edge surgical suture needle. The catheter was advanced 6–8 cm downstream and tied to the vessel wall by a pursestring ligature.

The cannulation presented no significant obstruction to venous flow since the vena cava cephalica is a large vessel of 8–10 mm diameter. Arterial blood was obtained by cannulation of the efferent branchial artery accessible at the lateral aspect of the gill when the mantle was relaxed. After tying a holding thread around the tip of the gill, a hole was cut in the efferent branchial vessel about 10–15 mm from the holding thread.

Polyethylene catheters (PE 50) were used except in one specimen where the highly contractile gill tissue precluded insertion of a catheter larger than PE 10. The catheters had a standard length of 40 cm and each blood sampling procedure lasted 20–30 s. The catheters were advanced 3–5 cm upstream from the point of insertion. A ligature was tied around the vessel immediately proximal to the point of cannulation.

This cannulating procedure occluded the distal portion of one of the two gills. The reduction in the overall gas exchange area from this occlusion can, however, only be small since the 40–60 cm long gills taper sharply off at the distal tips where the cannulations were made. Following cannulations the animals were transferred to well-aerated sea water and normally recovered within 10–20 min. No blood sampling or other experimentation was done until the following day, when the animals were assumed to have fully recovered from the cannulating procedures.

During experiments, arterial and venous blood were drawn simultaneously. The samples were kept in stoppered syringes in iced water until analysed for and pH using Radiometer equipment. Total blood CO₂ content was analysed on a modified Van Slyke apparatus (Brix, 1981). During hypoxia experiments, water was brought down to, and kept at, the desired level by nitrogen bubbling. Information on blood respiratory properties used in calculations was taken from Brix, Lykkeboe & Johansen (1981) and unpublished information. O₂ capacity was calculated as half of the blood copper content (Ghiretti, 1966) measured on a Perkin Elmer 503 atomic absorption spectrophotometer. All experiments were performed at 17–19 °C.

Table 1 gives values for arterial and venous and pH from steady-state conditions in normoxic (133 mmHg < < 153 mmHg) and hypoxic water (30 < < 8ommHg). In normoxic water the arterial was very high, exceeding 100 mmHg for 4 out of 5 animals. In two animals arterial was higher than no mmHg, in one case 125 mmHg, when ambient water was 135 mmHg. This implies near equilibrium between arterial blood and inspired water with respect to O₂ tension. Arterial blood was nearly fully saturated with O₂ (Table 1). Mixed venous in normoxic animals ranged between 17 and 40 mmHg. Table 1 shows the venous O₂ saturations to vary considerably, because of variable blood pH and the very large Bohr factor. The utilization or turnover of O₂ in arterial blood to the tissue also varied and could be as high as 99%. The blood O₂ capacity averaged 3·1 vol%. In 4 animals, total blood CO₂ content was measured in arterial and venous samples at normoxic conditions. The increases in blood CO₂ content (in mM), comparing arterial and venous blood for the 4 specimens, were from 2·7 to 4·5, from 3·3 to 5·0, from 3·0 to 4·6, and from 4·7 to 5·4.

During hypoxic exposure the arterial 0₂ tension fell markedly (Table 1, Fig. 2), while the venous tension fell initially, but as the hypoxia progressed it realigned with pre-hypoxic values. Blood pH rose markedly during hypoxia, also correlated with a state of hyperventilation. In some animals under hypoxic conditions, pH of venous blood was higher than that of arterial blood, although this finding was not consistent in all sample sets (Table i). Hypoxia also caused the total CO₂ content of arterial blood to rise (Fig. 1). The O₂ saturation fell in both arterial and venous blood, but the relative decline of the two, and thus of the O₂ utilization, varied between animals and experiments.

Fig. 1 (lower panel) also shows that O₂ utilization from circulating blood initially rises during hypoxic exposure (at P₁ ≃ 80 mmHg), but declines at more severe O₂ deficiency in the water.

Fig. 2 shows how the arterial and venous O₂ tensions relate to ambient during hypoxic exposure. The decline in arterial O₂ tension with reduced ambient was associated with a reduced gradient from arterial blood to inspired water. This trend was correlated with a marked hyperventilation. The venous O₂ tensions stayed rather unchanged, after an initial drop in moderately hypoxic water. In combination with a marked alkalosis, this implies an increase in venous O₂ saturation and hence a reduced utilization of circulating O₂ during hypoxia (Fig. 2, Table 1).

Fig. 3 shows the relationship between blood O₂ affinity (P₅₀), HcO₂ saturation and blood ⁠, based on O₂-binding curves determined for Sepia in vitro (Brix et al. 1981). The relationship between pH and O₂ affinity is expressed by the equation: log P₅₀ = −1·6 pH+ 13·644 at a temperature of 17 °C. The empirically determined n-value for the O₂ binding curves was 4·7. The broken lines show calculated isopleths based on an n-value of 2·5. The pairs of arterial and venous sample sets plotted on to the nomogram (larger data points) express the in vivo blood gas levels from unrestrained Sepia during normoxia (data set I) and during progressive hypoxia (set II, 80 mmHg; set III, 67 mmHg; and set IV, 30 mmHg). The smaller data points connected by arrows to those experimentally determined show what effect a reduction in n-value would have on arterial and venous O₂ saturations. It is clear that, for some data sets, the venous values are aligned with or displaced to the left of the arterial, implying that venous blood had a higher O₂ affinity, i.e. lower P₅₀ values than arterial. This result reflects the fact that circulating blood pH in some cases was higher in venous compared to arterial blood (Table 1). At a water of 67 and 30 mmHg, arterial and venous blood pH, and hence the corresponding O₂ affinity, were nearly similar.

Fig. 3 also reveals that a reduction in n-value from the very high level present in Sepia blood, results in a marked reduction in arterial saturation and a concurrent increase in the venous saturation, causing a drastic fall in the arteriovenous O₂ content difference and hence in utilization of the circulating blood O₂.

The combination of high tissue O₂ requirement and low blood O₂ carrying capacity such as is the case for cephalopods (Johansen et al. 1981 ; Ghiretti, 1966), place special demands on efficiency in utilizing the oxygen transport potential of the blood. This in turn will depend on the respiratory properties of blood and the ventilatory and circulatory pumps, as well as the diffusion barriers in the gills and tissues. A high blood O₂ utilization expressed by a near-maximal difference in O₂ content between arterial and venous blood has long been advocated as a unique trait of cephalopod blood and alleged to depend on an exceptionally high pH sensitivity (Bohr effect) of the binding of O₂ to most cephalopod haemocyanins. Recently, however, Lykkeboe, Brix & Johansen (1980), Brix et al. (1981) and Lykkeboe & Johansen (1982) have pointed out that a Bohr factor (Δ logP₅₀/ΔpH) numerically exceeding 1·0, which is typical of many cephalopods, brings about an entirely different interaction between blood acid-base status and oxygenation of the haemocyanin than when the Bohr factor is numerically less than 1·0. The important point emphasized in these recent studies is that the maximal yield of protons from aerobic metabolism will numerically match that of oxygen used in metabolism on a molar basis, if the gas exchange ratio, R_E= 1·0. It should be remembered that the binding of O₂ to haemocyanin is tanta-mount to the release of the same mol fraction protons, or, conversely, during O₂ unloading one mol protons will be bound to the O₂ carrier when one mol O₂ is unloaded. This relationship formally expressed in the linkage equation (Wyman, 1964) has the important consequence that if the pH sensitivity of the blood numerically exceeds 1·0, there will not be an excess of free protons produced in aerobic metabolism to shift the O₂-binding equilibrium of haemocyanin towards that of unbound O₂, i.e. O₂ unloading in the tissues will consequently not be promoted by the aerobic metabolism. Rather, as predicted by Lykkeboe et al. (1980), the pH of venous blood may exceed that of arterial blood, handicapping O₂ unloading rather than promoting it as has been traditionally advocated (Redfield & Goodkind, 1929).

The present study on Sepia is one of very few allowing an evaluation of the importance of the uniquely high Bohr shifts in cephalopods for blood gas transport in vivo based on blood samples drawn from unrestrained specimens.

The high arterial O₂ tensions in Sepia (Table 1, Fig. 1) at normoxic ambient conditions, suggest a highly efficient gas exchange in the gills. An arterial of 100 mmHg implies that an O₂ extraction from the ventilatory current of 28·5% or higher will suffice to produce a negative gradient between expired water and arterial blood, characteristic of a counter-current exchange process. Hazelhoff (1939) reported a range for O₂ extraction between 50% and 80% in cephalopods, while the large Octopus dofleini showed an average extraction of 26-8% (Johansen & Lenfant, 1966).

Arterial blood for Sepia in well-aerated water will therefore typically be fully saturated with O₂ (Table 1). The more variable venous corresponds to venous O₂ saturations that are about 20% or lower, implying O₂ utilizations 80% or higher. At conditions of increased O₂ requirement during swimming, or at reduced water ⁠, the cephalopod solution to increased or maintained O₂ delivery (hypoxic water) can thus only modestly depend on an increased unloading of O₂, but must instead depend on maximizing the arterial O₂ saturation and an increase in cardiac output.

Our data testify that in Sepia venous pH may exceed arterial pH, in both normoxic and hypoxic water (Table 1). This in vivo situation accords with the predictions of Lykkeboe et al. (1980), explainable as a result of the interaction between proton binding and the state of oxygenation of haemocyanins with very high Bohr shifts. As is apparent from Fig. 3, this tendency gives venous blood a higher O₂ affinity than arterial blood, a fact which will actually impede O₂ unloading from haemocyanin.

In addition, the O₂ affinity of all circulating blood will increase during hypoxia as a result of a general alkalosis, a finding recently also reported for blood of Octopus vulgaris (Houlihan et al. 1982). This tendency will further reduce O₂ unloading, while at the same time favouring O₂ loading. The possibility that anaerobic production of protons could aid O₂ unloading, for example during exercise, is small due to the relatively undissociated state of octopine, which is the principal product of anaerobic metabolism in cephalopods (Zammit, 1978).

The very high O₂ utilization of Sepia blood in normoxic and moderately hypoxic water (⁠ ≅ 80 mmHg) is clearly related to the highly sigmoid shape (high n-value) of the O₂HC binding curves (Fig. 3). Calculated data points show that arterial saturations would decline and the venous saturations markedly rise, if the blood had a smaller n-value. The high n-value can hence be regarded as a counterbalance for the apparent adverse influence of the blood acid-base status on O₂ unloading. Fig. 3 (top panel) also gives the numerical changes in arteriovenous O₂ content difference, O₂ utilization and the perfusion requirement ⁠. The latter is seen to increase dramatically as the blood loses importance in O₂ transport.

Accompanying the alkalosis in Sepia during exposure to hypoxia was a notable increase in total CO₂ content of the blood (Fig. 1). The increase may possibly be related to the breakdown of arginine phosphate that occurs during hypoxia (Storey & Storey, 1979), since hydrolysis of this compound leads to a build-up of bicarbonate (Burton, 1978). The phenomenon will act to amplify the tendency towards an alkalotic status during hypoxia and thus further substantiate the O₂ loading strategy of compensatory O₂ transport.

We are grateful to the Marine Biological Association of the United Kingdom at Plymouth, England, for help with a supply of Sepia, and for space and research facilities. This investigation was supported by a NATO grant to Drs P. W. Hochachka and K. Johansen, Dr O. Brix was supported by the Danish Natural Science Research Council, J. nr. 511-20410.