Although the results we have recorded merely serve to indicate the possibilities of this interesting field of investigation, we have sufficient data to enable us to draw certain general conclusions. In the first place it is evident that the bloods of the more highly developed marine invertebrates, such as the active Crustacia and the Cephalopods, are specially adapted for the carriage of carbon dioxide. The quantity of carbon dioxide taken up by the blood of Maia, Palinurus, or Octopus at any given tension of the gas is, in general, about twice or three times as great as that which is taken up by sea water under the same conditions. On the other hand, the blood of a slow, creeping form, such as Aplysia, or of a sessile animal such as the ascidian Phallusia shows no more adaptation for the carriage of carbon dioxide than does sea water.
But our estimations of the CO2 content of the blood as it circulates in the bodies of these more active invertebrates show that the conditions of transport of this gas differ considerably in some respects from those which obtain in mammals. For the invertebrate blood in the body contains only a relatively small quantity of carbon dioxide, averaging in the forms we examined from 3 to 10 cc. per 100 cc. of blood. This forms a marked contrast with the condition found in mammals where even the arterial blood contains about 50 cc. of CO2 per 100 cc. of blood. The invertebrate, therefore, works at a very low CO2 tension. There is a twofold significance in this circumstance. In the first place, it means that only the first portion of the carbon dioxide dissociation curve is in use in the respiratory mechanism. Now an inspection of our curves will show that at these low carbon dioxide tensions the dissociation curves tend to be steeper than at higher tensions. As we intend to show in a later paper it can be proved mathematically that, other things being equal, a blood with a carbon dissociation curve of moderate steepness, i.e. one in which the carbon dioxide content of the blood increases fairly rapidly with increase of carbon dioxide tension, is a more efficient carrier of the gas from the tissues to a respiratory surface than a blood in which the dissociation curve is either steeper or flatter. It would seem as if the active invertebrates avoid the use of too flat a part of their CO2 dissociation curves by working over the initial steeper portion.
Furthermore, it is seen that over the range of this initial steep portion of the curves the changes of reaction produced by the uptake of carbon dioxide are much smaller than at higher tensions of the gas; for these initial portions of the curves are more nearly parallel to the lines of constant reaction calculated for a temperature of 15°C. according to Hasselbalch's method (10) on the assumption that the whole of the combined CO2 is in the form of sodium bicarbonate. It is evident also that on this assumption the hydrogen ion concentration of the blood of invertebrates (with the exception of the tunicates) would appear to be practically the same as that of the warm-blooded vertebrates—a conclusion confirmed by the direct measurements of Quagliariello (9). On the other hand, our measurements do not lend support to the idea put forward by Collip (4) that in order to maintain an appropriate faintly alkaline reaction an invertebrate needs to retain carbon dioxide in its blood at a comparatively high tension. This idea was based on the observation that at comparatively high CO2 tensions the blood of invertebrates contains considerably more sodium bicarbonate than does sea water. But our curves show that this is no longer true at the lower values of carbon dioxide tension, the amount of sodium bicarbonate falling off more rapidly in the blood than in the sea water with diminution of the carbon dioxide tension so that in order to maintain an appropriate reaction in the blood only a comparatively small tension of CO2 is required. The largest amount of carbon dioxide that we found present in the circulating blood of any of the types examined was 9.7 cc. per 100 cc. of blood in the case of Maia, and in most cases the amount was considerably less. But even this lowest value corresponds to a tension of CO2 of only about 3 mm., so that the tension gradient across the gill membrane must be even less than this. We would emphasize rather the circumstances that as the portion of the dissociation curve over which the reaction is approximately constant is of but small extent, it is necessary that in an active form like Octopus the carbon dioxide produced should be removed rapidly lest an accumulation of it should cause the limits of normal reaction to be exceeded; and this need is correlated with the extreme efficiency of the respiratory apparatus in this animal. It is interesting to notice that the mammal which, in order to obtain an appropriate reaction in the blood, has to work at relatively high carbon dioxide tensions where the dissociation curve is comparatively flat, secures a steeper physiological CO2 dissociation curve in the body, and with it a more efficient carriage of carbon dioxide and a more constant reaction in the circulating fluid, in virtue of the effect of oxygenation on the carbon dioxide-combining power of its blood (3, 6).
Returning now to the consideration of the actual form of the dissociation curves we have obtained—it is a significant fact that it is in those forms such as Maia, Palinurus, and Octopus whose bloods are rich in proteins—particularly hemocyanine—that the initial steep portion of the curve is observed. This suggests that in these forms the blood proteins act as weak acids and expel carbon dioxide from the blood at the low tensions which include the physiological range, just as in vertebrates the hemoglobin similarly displaces carbonic acid from its combination with alkali metal. On the other hand the cœlomic fluid of Aplysia contains no pigment and only 0.00672 per cent of protein nitrogen (Bottazzi (11)) and shows no initial rapidly ascending portion of the CO2 dissociation curve. This is supported by the observation of Quagliariello (9) that the acid-neutralising power of the blood of an invertebrate is roughly proportional to its protein content. It seems as if the proteins of invertebrate blood like the blood proteins of vertebrates, exist in the form of sodium salts which are capable of giving up sodium for the transport of carbon dioxide as sodium bicarbonate. That this is so in the case of hemocyanine follows from the fact that the isoelectric point of this pigment occurs at a hydrogen ion concentration of 2.12 x 10–5 N, i.e. at a pH of 4.67 (Quagliariello (12)) so that in the alkaline blood of the invertebrates possessing it, hemocyanine will act as a weak acid. It is probable that the initial steep portion of the carbon dioxide dissociation curves which we have found to be of such importance in the respiration physiology of Octopus, Palinurus, and Maia is produced by the competition of this acid with carbonic acid for the available sodium of the blood.