Studies on the Physiology of Ascaris Lumbricoides: II. the Inorganic Composition of the Body Fluid in Relation to That of the Environment

The observations of Hobson et al. (1951) show clearly that the body fluid of A. lumbricoides changes appreciably in composition when the animals are exposed to a purely saline medium such as 30% sea water, and that its chloride content is considerably increased. Although no artificial medium lacking organic constituents corresponding to those occurring in the normal environment can be quite satis-factory, it is clearly desirable that its electrolyte composition should correspond as closely as possible to that of the intestinal fluid of the host. Further, the relation of the composition of the external medium to that of the body fluid of the worm should be considered.

For this reason analyses have been made of the principal inorganic constituents of the intestinal fluid of the pig, of some artificial media, of the body fluid of fresh worms and of worms which have been exposed to the saline media. These were carried out by A. Eden at the Ministry of Agriculture and Fisheries’ Veterinary Laboratory, Weybridge. W. Stephenson and A. D. Hobson were responsible for the conductivity and osmotic pressure determinations as well as for some of the measurements of chloride concentration.

The samples of the various body fluids were taken to Newcastle in test-tubes, stoppered with rubber bungs, and packed in large thermos flasks containing cottonwool and broken ice. They were dispatched by passenger train to Weybridge, and the temperature of the samples on receipt was always below 5° C. Each sample was warmed to 20° C., and a known volume (5 or 10 ml.) was weighed into a silica basin to obtain the specific gravity. Most of the moisture was then carefully evaporated off by placing the basin on a hot plate, and the residue was then dried to constant weight in an oven at 105 ° C. The basin was ignited gently, first over a small flame and then in a muffle furnace at 500° C., until all carbonaceous matter had burned away, giving the total ash figure.

The ash was taken up carefully with dilute HC1, evaporated to dryness, and dissolved in hot water in the presence of 1 ml. decinormal HC1. The resultant solution was washed into a 200 ml. flask, and the transference completed by hot water ; after cooling the contents of the flask were made up to the mark and thoroughly mixed. This solution was used for the determination of calcium, magnesium, sodium and potassium.

Calcium was determined on a suitable aliquot (1, 2 or 5 ml.) by the Clark-Collip (1925) modification of the Kramer-Tisdall procedure, being precipitated as oxalate, which was determined by acid-KMnO₄ titration. The supernatant fluid of the original precipitation, obtained by centrifuging the oxalate precipitate, was used for the estimation of magnesium, which was precipitated as magnesium ammonium phosphate and the latter determined by the molybdenum blue procedure of Fiske & Subba Row (1925) for inorganic phosphate. Sodium was determined gravimetrically by precipitation in a sintered glass crucible with zinc uranyl acetate, after removal of phosphates with solid calcium hydroxide. Potassium was determined on suitable aliquots by sodium cobaltinitrite precipitation by a modification of the JacobsHoffman procedure (Eden, 1943).

Phosphorus was determined separately on 2 ml. portions of the original body fluid by wet digestion to remove organic matter employing sulphuric, nitric and perchloric acids, dilution to a known volume, and developing the molybdenum blue complex by the method of Fiske & Subba Row (1925). This procedure gave a total phosphorus figure, since it was felt that inorganic phosphorus determination might give unreliable results, owing to the uncertain effect of any phosphatases that might be present. Chlorides were also determined on the original fluid by a minor modification of the Whitehorn method (1921) for blood.

A. lumbricoides normally inhabits the small intestine of its host. In the investigations described here the animals were obtained from the pig. In Table 2 are given the results of analyses of a number of samples of the intestinal contents of each pig. Each sample was composed of approximately equal volumes of fluid obtained by centrifuging material obtained from the small intestine of a number of pigs immediately after slaughtering.

It will be noted that the samples fall into two groups which differ considerably in their electrolyte composition. Samples A and B, taken on the same day, are characterized by the fact that their sodium, potassium and magnesium contents are about twice as great as those of samples 31-36, also taken on one day. The volumes for calcium, on the other hand, are of the same order of magnitude in both sets if we except the extraordinarily high concentrations in samples 31 and 35. It should be noted that the values given for phosphorus do not distinguish between inorganic and organic phosphorus.

The analyses quoted show that there is, as might perhaps be expected, a great variation in the electrolyte composition of the medium to which the worms are normally exposed. The causes of these differences cannot be determined without detailed knowledge of the previous history of the pigs from which the material was obtained. This was not available.

Little has been published on the composition of the body fluid of nematode worms. Flury (1912), Schopfer (1924, 1926) and Duval & Courtois (1928) give values for the chloride concentration in Par ascaris equorum. Rogers (1945) made determinations of certain constituents of the body fluid of Ascaris lumbricoides, and average values calculated from his figures are quoted at the foot of Table 2. He also gives data for glucose and for protein and non-protein nitrogen.

In Table 3 are given the results of the analyses of seven pooled samples of the body fluid of varying numbers of worms which had been taken direct from the host and had not been exposed to any artificial saline medium. These show that the electrolyte composition of the body fluid is fairly constant, especially in its calcium and magnesium content. At the risk of being accused of selecting figures it may be permissible to compare the mean values for the body fluid with those for samples 31-36 of the intestinal fluid of the pig, leaving out the calcium figures for samples 31 and 35, which are obviously abnormal. This comparison is given in Table 4.

From these figures it appears that the electrolyte composition of the body fluid of A. lumbricoides is closely similar to that of its normal environment, with the exceptions that the phosphorus and chloride concentrations are lower. The problem of the chloride concentration has been discussed in an earlier paper (Hobson et al. 1951).

In earlier work (Hobson et al. 1951) 30% sea water was used as a balanced saline medium for keeping animals alive in the laboratory. This proved satisfactory for short-term experiments, and individuals have been kept for as long as 28 days alive and healthy as far as could be judged by external appearance and activity. It seemed, however, important to discover what changes occur in the electrolyte composition of the body fluid of worms exposed to this and other saline media. The values after various times of exposure are given in Table 5.

The results may be summarized as follows:

The sodium concentrations of the three saline media used did not differ greatly from those of the intestinal fluid of the pig as shown by the analyses of samples 31-36 in Table 2. The amount of sodium in samples A and B seems to be abnormally high. The sodium concentration of the body fluid of Ascaris, although somewhat variable, is of the same order of magiïitude in fresh animals and in those which have been exposed to saline media. The data presented do not indicate that the concentration of sodium in the body fluid of the animals varies with that of the external medium. It must be remembered, however, that the range of concentrations in these experiments was small.

Comparison of the values given in Tables 2 and 3 suggests that the concentration of potassium in the body fluid of fresh Ascaris, although variable, is approximately the same as that of its normal environment. (The potassium content of samples A and B of intestinal fluid seems, like that of the sodium, to be abnormally high.) When the animals are transferred to a saline medium there is a marked fall in the potassium concentration of the body fluid. Moreover, the internal concentration does not show any clear correspondence with that of the external medium.

The concentrations of these two elements in the body fluid of Ascaris remain very constant, and there is no significant difference between the values obtained with fresh worms and those which have been exposed to artificial media.

In an earlier paper (Hobson et al. 1951) it has been shown that the chloride concentration of the body fluid varies with but always remains below that of the external medium. This conclusion receives further support from the data presented here.

The results obtained in this investigation are admittedly not as extensive as is desirable but they allow certain conclusions to be drawn.

1. The sodium and potassium contents of the body fluid of Ascaris lumbricoides are somewhat variable, but these variations do not seem to be dependent upon those of the external medium.

2. The calcium and magnesium contents of the body fluid are relatively constant and are not affected by those of the external medium.

3. The chloride concentration of the body fluid is closely related to and always remains lower than that of the external medium.

4. As shown in Table 2, there is a large gap between the total concentrations of inorganic cations and anions in the intestinal fluid of the pig. Presumably a considerable proportion of the inorganic cations are combined with organic anions, at present undetermined. Exposing the worms to saline media composed of chloride caused a large rise in the internal chloride concentration. This may well be a limiting factor in the life of the animals in such media, and the next step forward would seem to be the fuller analysis of the environment to which they are normally exposed.

We are greatly indebted to the Agricultural Research Council and to the Research Committee of King’s College for financial support of these investigations. We are also extremely grateful to Dr H. H. Green for affording facilities to carry out the analyses in the Biochemical Department of the Veterinary Laboratory of the Ministry of Agriculture and Fisheries at Weybridge.