Carbonic Anhydrase Activity During Embryonic Development

The enzyme carbonic anhydrase, which catalyses the reaction CO₂+H₂O ⥦H₂CO₃, is known to play an important part in the transport of carbon dioxide in the blood of higher vertebrates (Meldrum & Roughton, 1933; Roughton, 1935, 1943; Keilin & Mann, 1941). It is also concerned in the regulation of disturbances in acid-base equilibrium associated with the production of large quantities of hydrochloric acid by the gastric mucosa (Davies, 1948). As it is becoming increasingly evident that this widely distributed enzyme is one of the most important single factors enabling rapid adjustments to be made in acid-base regulation, it is desirable that we should have some knowledge of its formation and physiological importance during embryonic development.

Meldrum & Roughton (1933) observed that carbonic anhydrase does not appear in the circulating blood of the goat foetus until just prior to birth. In human blood taken from the umbilical cord immediately after birth, the enzyme activity is only about one-half that of adult blood (Van Goor, 1934), while still lower values may be observed for the bloods of prematurely born infants (Stevenson, 1943). Van Goor (1940) has reported that there is no carbonic anhydrase in the blood of the developing chick until the twelfth day of incubation, although the enzyme is present in the optic vesicles as early as the third day. Ashby (1943) found very low activity in the blood of the foetal rat, but in a more recent paper (Ashby & Butler, 1948) it is stated that foetal cattle appear to be exceptional in that the blood contains a relatively high concentration of the enzyme.

Van Goor’s observation that the enzyme is present at such an early stage in the formation of the eye indicates that carbonic anhydrase is probably synthesized more or less independently in each of the tissues in which it occurs in the adult. Although some observations were made on the carbonic anhydrase activity of several embryonic organs, the chief aim of the present investigation was to ascertain whether the changes in the site of formation of the blood cells bring about any corresponding variations in the amount of enzyme contained within the erythrocytes. The specific points considered, therefore, are the following. At what stage in development is carbonic anhydrase formed in the blood? Is it present in both primitive and definitive series of red cells? In which haematopoietic tissues is it formed and what changes in activity take place in relation to increase in body size and in the haemoglobin content of the blood? The observations described below enable answers to be given to these questions.

Carbonic anhydrase activity was determined manometrically at 0°C. by the boat method of Meldrum & Roughton. One side of the boat contained 2·0 ml. of 0·186 M-sodium bicarbonate dissolved in 0·038 M-sodium hydroxide (Hodgson, 1936), while the other contained 1·0 ml. of 0·4M-phosphate buffer of pH 6·8 and distilled water or enzyme solution to make up a final fluid volume of 4·0 ml. The enzyme unit adopted was that amount of carbonic anhydrase which doubled the initial rate of evolution of carbon dioxide, in /d. per sec., at 0°C., pH 6·8 and a shaking rate of 350 cyc./sec. Precautions were taken to ensure that diffusion did not constitute a limiting factor (Clark, 1949).

Haemoglobin was estimated by Szigeti’s method (1940), using the Hilger-Spekker absorptiometer. When the concentration of the pigment was very low, as for example in the homogenates, the more sensitive pyridine haemochromogen method was used in conjunction with a micro-spectroscope and double-wedge trough (Elliott & Keilin, 1933). Blood from chick embryos and foetal mice was taken from the heart or umbilical vessels by means of a capillary pipette. All homogenates were prepared in distilled water.

To enable comparisons to be made between the activities of blood samples containing different numbers of corpuscles, two ratios were calculated. The first, A/Hb, is the number of enzyme units per µl. of whole blood divided by the haemoglobin concentration of the blood in grams per cent. Any change in either the amount of carbonic anhydrase in the red cell or in the corpuscular haemoglobin may affect the value of A/Hb. The second ratio, CA, is the number of enzyme units per million red cells and can be used to distinguish between variations which are actually due to changes in the amount of enzyme contained within the red cells and those merely due to differences in the red cell count.

Confirming Van Goor’s observation, the blood of the developing chick embryo was found to contain no carbonic anhydrase until about the twelfth day of incubation. On the fourteenth day the enzyme is present in appreciable amounts and increases rapidly until the adult level is reached by the eighteenth day. The changes in the haemoglobin content of the blood and in the red cell count which occur during this period are shown in Figure 1, each point representing the mean of three observations. The cell count increases enormously between the fifteenth and nineteenth days but there is a marked fall just before hatching. This increase is reflected by a similar increase in the carbonic anhydrase activity. If, however, we consider not the apparent enzyme activity of the blood but the ratio A/Hb, then it is evident (Fig. 2) that the formation of carbonic anhydrase in relation to haemoglobin follows a smooth sigmoid curve. The ratio between the two proteins which is characteristic of the blood of the adult is not reached until after hatching.

The physiological significance of the increase in carbonic anhydrase activity during the last period of development has been pointed out by Van Goor and by Needham (1942). It is of interest to note that during this period there is a sharp rise in the carbon dioxide output curve for the whole egg (Noyons & De Hasselle, 1939).

Having established the time at which the enzyme appears in the blood, the next point to consider is whether it is confined to any particular series of erythrocytes or whether it is present in all of them. Fig. 3 shows the changes which take place in the value of CA as development proceeds. It is clear that the enzyme must be entirely absent from the primitive line of red corpuscles, for there is little or no production of these cells after the fifth day, although according to Dawson (1936) a few may persist in the circulation until 2 weeks after hatching. They represent only a very small fraction of the total red cells at the time when carbonic anhydrase activity is first observed in the blood. The conclusion must be, therefore, that the blood islands, blastoderm and endothelial lining of the vascular system are not regions where carbonic anhydrase is formed.

The first of the definitive cells likewise appear to lack the enzyme, for they already make up the greater portion of the red cell population by the sixth day. From this time onwards, there is a steadily increasing degree of haemopoietic activity, first of all in the spleen, then in the walls of the yolk sac and finally in the bone marrow. The curve in Fig. 3 shows that the onset of blood formation in the marrow coincides with the increase in carbonic anhydrase content of the corpuscles, although the participation of the other organs is not entirely excluded for there is considerable overlapping. The yolk sac in particular is quite active until about the fourteenth day. However, the fact that the blood-forming activity of the bone marrow increases at the same time as the enzyme activity of the blood, while during the same period the other organs show a gradual decline in their haemopoietic functions, does seem to favour the interpretation that the relatively sudden appearance of carbonic anhydrase in the blood is due to the onset of haemopoiesis in the bone marrow. It is therefore of some interest to consider whether bone marrow itself shows any enzyme activity.

The femurs of advanced embryos (15−21 days) were removed and split longitudinally. The marrow from one bone was homogenized and used for the estimations of carbonic anhydrase and haemoglobin. The corresponding femur from the opposite side was placed in a small tube containing a known volume of diluting fluid and the marrow scraped out as completely as possible. The bone was transferred to a second tube of fluid and washed for several minutes, the two portions of fluid then being combined quantitatively. This was readily accomplished by the use of wax-coated glassware. The suspension was mixed thoroughly and the number of cells present counted with a haemocytometer. Smears were also made and the proportions of mature and immature red cells determined.

Some typical results are shown in Table 1. There is an enormous increase in the haemopoietic activity of the marrow just before and immediately after hatching. On the other hand, there is a definite fall in the red cell count of the circulating blood during this period. As it is not very probable that the total blood volume rises very much between the nineteenth and twenty-first days, this can only mean that there is an active replacement of erythrocytes at the time of hatching.

It may also be noted from Table 1 that the values of A/Hb for bone marrow are always much higher than the corresponding values for the circulating blood. This finds its explanation in the fact that very high values of A/Hb are characteristic of immature erythrocytes, a point that may be demonstrated by increasing the proportion of immature cells in the circulation through severe haemorrhage (Van Goor, 1943 ; Clark, unpublished). It is not yet clear whether the enzyme is synthesized within the corpuscle before haemoglobin, or whether it passes through a maximum as the cell matures. Either possibility could account for a high value of A/Hb. If, on the other hand, the haemoglobin is being formed in the absence of carbonic anhydrase, then A/Hb will tend to be very low. Comparison of the ratios for blood, bone marrow and yolk sac thus suggests that the latter produces red corpuscles which are devoid of the enzyme (Table 2). It may be argued that the comparison of values of A/Hb for blood with those for homogenates is not valid, in so far as the first case concerns a population of cells more or less identical, whereas the tissues contain not only erythrocytes but granular leucocytes in various stages of differentiation, as well as the endothelial cells lining the blood sinusoids. This objection is not as serious as might at first appear, for not only do the erythroblasts and erythrocytes greatly outnumber the other cell types present, but they are also the only ones known to possess very high carbonic anhydrase activity. Leucocytes do not contain the enzyme, even though they appear to have a relatively high zinc content (Vallee & Gibson, 1948). It is reasonable to conclude, therefore, that the carbonic anhydrase activity of bone marrow is due almost entirely to the red cells present Owing to the greater difficulty in obtaining sufficient blood, the observations on developing mice are not as complete as those on chicks. Nevertheless, a fairly clear picture can be presented of the main changes occurring during development. No carbonic anhydrase appears in the foetal blood until about the fifteenth day (Table 3).

The actual value of A/Hb or of CA at any given stage is subject to considerable variation, even within litter-mates, but this variability tends to be reduced in older foetuses. It may be due in part to small individual differences in the times at which the various haemopoietic tissues assume functional importance.

Contrary to the situation in the chick, the carbonic anhydrase activity of mouse blood does not reach maximum levels until several weeks after birth (Fig. 4). This is presumably a reflexion of the comparatively short gestation period. The relative slowness with which the various haematological characteristics approach steady values (Table 4) suggests that the replacement of red corpuscles, which is believed to take place at birth in the mammal (Smith, 1932; Wintrobe & Shumacher, 1937), proceeds rather slowly in this species.

The only published observations on the carbonic anhydrase activity of embryonic tissues are those of Van Goor (1940) and of Ashby & Butler (1948), both of which deal more particularly with parts of the nervous system. It has already been mentioned that Van Goor found the enzyme to be present in the optic cup of the 72 hr. chick. Ashby & Butler, working on foetal cattle and prematurely born human infants, find that in the central nervous system the adult pattern of distribution of the enzyme is reached by the beginning of the last quarter of gestation. The cerebrum appears to be exceptional in that no carbonic anhydrase activity is apparent until just before birth, in the case of cattle, and not until after birth in the case of humans. In view of its relatively late appearance in the developing central nervous system, carbonic anhydrase differs from other enzymes that have been investigated. Succinic dehydrogenase, cytochrome oxidase and choline esterase, for example, all appear at an early stage, the rise in their activities being closely associated with increasing morphological complexity (Nachmansohn, 1940; Youngstrom, 1941; Flexner, Flexner & Straus, 1941, 1946). These enzymes are probably of far greater importance for the metabolism and functional activity of nervous tissues. It may be noted that Davenport (1946) has shown that carbonic anhydrase plays no essential role in the activity of nerve fibres.

In the present investigation, the organs selected for study were the lens, retina, stomach, kidney and brain. Corrections for the enzyme activity of traces of blood present in the homogenates were made by measuring the concentration of haemo-globin, using the pyridine haemochromogen method, and calculating the value of A/Hb for both tissue and blood.

Carbonic anhydrase develops more or less independently in the different organs and at quite different rates (Fig. 5). Only in the stomach does the increasing activity occur at about the same time as that observed in the blood. However, the most striking feature is the way in which the enzyme may be formed in the organs long before the onset of functional activity. As Barcroft (1934) remarked in another connexion, ‘the stage is set before the play commences’. The retina is the extreme case of this. Bakker (1939) and Leiner (1940) have suggested that the high rate of glycolysis of the vertebrate retina necessitates the presence of a large amount of carbonic anhydrase to facilitate the removal of carbon dioxide, but there would appear to be no real evidence to support this, since it has yet to be shown that the non-enzymically catalysed rate for the reaction H₂O + CO₂⥦H₂CO₃ at 38°C. is inadequate for physiological requirements. In any case, it is perhaps open to doubt whether the presence of the enzyme in large amount would in fact facilitate the removal of carbon dioxide, for it would convert rapidly diffusing gas molecules into the more slowly diffusing bicarbonate ions.

It is rather difficult to assess the amount of functional activity which takes place in the other organs before birth. According to Gundobin (1912), as soon as gastric glands become recognizable histologically they can secrete acid, but this has been denied by Schmidt (1914), who claims that no acid is produced until after birth. Sutherland (1921), however, found convincing evidence for the secretion of acid in the foetal guinea-pig stomach, which suggests that gastric carbonic anhydrase is already playing a role in acid-base regulation (Davies, 1948). There is also evidence to show that a considerable amount of renal activity takes place before birth (Needham, 1942; McCance, 1948), in which the enzyme undoubtedly participates, but accurate information for either the chick or the mouse is lacking.

The changes which occur in the values of A/Hb and CA during the latter period of development in the chick embryo are of considerable interest in connexion with the work of Hall (1934). He found that from about the tenth day of incubation until 3 weeks after hatching there is a gradual shift in the dissociation curve of chick haemoglobin, indicating a decrease in the affinity of the pigment for oxygen. Hall explains this by supposing that an embryonic type of the pigment is slowly replaced by an adult type. As Dawson (1936) points out, this cannot be due to the replacement of the primitive red cells by those of the definitive line, for the shift in the dissociation curve is most marked between the twelfth and eighteenth days. On the other hand, it is significant that the shift coincides closely with the appearance of carbonic anhydrase activity in the blood and with the onset of haemopoiesis in the bone marrow. Dawson has suggested that the shift in the dissociation curve is due to the replacement of red cells formed in the yolk sac by those formed in the bone marrow. Now the values of A/Hb given in Table 2 indicate that the corpuscles originating in the yolk sac lack carbonic anhydrase. Therefore, if we accept Dawson’s suggestion that these cells contain a haemoglobin different to that produced in marrow, it is then possible to explain the fact that the rapid shift in the dissociation curve, the increase in carbonic anhydrase activity and the growing importance of bone marrow as a haemopoietic tissue all occur at the same time. The replacement of corpuscles which is assumed to take place (Table 1) would fit in perfectly with this picture. It may be recalled that Smith (1932) found evidence indicating that there is a complete change in the blood cell population of the rat at birth. The results of the present observations suggest that what actually occurs is the replacement of a large foetal type of corpuscle, containing the embryonic type of haemoglobin but no carbonic anhydrase, by a smaller type of cell in which the enzyme is present together with the adult haemoglobin. Recently, Jonxis (1948) has also suggested that foetal and adult haemoglobins do not occur together in the same corpuscle. He claims that in erythroblastis foetalis the corpuscles containing foetal haemoglobin are broken down preferentially, while those containing the adult type are more or less unaffected.-

Using the method of alkali denaturation, Brinkman & Jonxis (1936) have shown that the relative proportions of foetal and adult haemoglobins present in blood can be ascertained. If it could be demonstrated that the replacement of the foetal type of haemoglobin commences at the same time that carbonic anhydrase activity appears, it would be strong evidence that a new kind of erythrocyte is put into circulation at that stage of development. In any case, it is difficult to see how one can explain the gradual increase in CA and A/Hb over a period when the red cell count may show sudden changes, except in terms of a mixed population of cells in which active replacement is proceeding.

This work was carried out during the tenure of an 1851 Exhibition Science Research Scholarship. My thanks are due to Prof. D. Keilin, F.R.S., for his interest in the problem, and to Dr P. Tate for providing some of the material.