Sansom & Manston, 1963) and rats (Payne & Sansom, 1963). It appeared

Transcription

1 J. Physiol. (1964), 170, pp Printed in Great Britain THE RELATIVE TOXICITY IN RATS OF DISODIUM ETHYLENE DIAMINE TETRA-ACETATE, SODIUM OXALATE AND SODIUM CITRATE BY J. M. PAYNE AND B. F. SANSOM From the A.R.C. Institute for Research on Animal Diseases, Compton, Newbury, Berkshire (Received 29 August 1963) In previous work in this laboratory solutions of sodium oxalate or of disodium ethylene diamine tetra-acetate (disodium EDTA) were injected intravenously to induce experimental hypocalcaemia in cows (Payne, Sansom & Manston, 1963) and rats (Payne & Sansom, 1963). It appeared from experiments with cows that disodium EDTA induced hypocalcaemia more rapidly than sodium oxalate administered at an equivalent rate. Thus, given in sufficient quantity both agents induced symptoms, but with disodium EDTA these occurred when the plasma calcium concentration was at its lowest point, whereas with sodium oxalate they occurred when the plasma calcium concentration was returning to normal after the injection had ceased. The purpose of the present experiments was to investigate these differences. It was hoped that a study of the relative toxicity of disodium EDTA and sodium oxalate when administered intravenously to rats might reveal the nature of the mechanisms involved in the removal of calcium from the plasma. Sodium citrate was included in the investigation as another chelating agent of potential value for inducing hypocalcaemia. METHODS The rats used in these experiments were inbred hooded Norwegian or Wistar albino. All were in a good nutritional state. Preparation of disodium, EDTA, sodium oxalate and sodium citrate soutions. Disodium EDTA solution was prepared as previously described (Payne & Sansom, 1963), the stock solution being equivalent to 5 93 mg Ca/ml. Stock solutions of sodium oxalate and sodium citrate of theoretically equivalent calcium-precipitating or chelating power were also prepared. Where necessary dilutions were made with NaCl solution 9 g/100 ml. Preparation of sodium oxalate containing freshly precipitated calcium oxalate and calcium phosphate. A precipitate of calcium oxalate was obtained by adding an excess of calcium chloride to 10 ml. of the stock solution of sodium oxalate. This was centrifuged and washed once with distilled water and once with stock sodium oxalate solution. It was then resuspended in either 100 ml. or 1 1. of stock sodium oxalate solution. These fluids therefore contained sodium oxalate equivalent to 5 93 mg Ca/ml. with the addition of calcium as calcium oxalate mg or mg/ml., most of which was in suspension. For the

2 614 J. M. PA YNTE AND B. F. SANSOM purpose of this paper they wiu be called sodium oxalate (CaOX 0-1) and sodium oxalate (CaOX 0-01) respectively. Suspensions of calcium phosphate in sodium oxalate solutions were prepared similarly. Calcium phosphate was precipitated from 10 ml. of a solution of calcium chloride containing 5 93 mg calcium/ml. with excess sodium orthophosphate. The precipitate was washed and resuspended in either 100 ml. or 1 1. of stock sodium oxalate solution. These solutions, which contained calcium as calcium phosphate either mg or mg/ml., will be called sodium oxalate (Ca3(Po4)2 0-1) and sodium oxalate (Ca3(Po4)2 0.01) respectively. Injection8. Ether anaesthesia was used throughout and all injections were made into the tail vein. Two techniques were used: (1) single injections in which the dose was administered at a steady rate over a period of 5-10 sec, or (2) chronic infusions in which the dose was administered at the rate of 0-05 ml. each 20 sec over a total period of 2-80 min. Special types of injection were used where mentioned in the text. (1) Freshly prepared suspensions of calcium oxalate or calcium phosphate in stock sodium oxalate were thoroughly shaken immediately before use and injected intravenously over a period of 5-10 sec. (2) The following procedure was used to investigate the rate at which the calcium oxalate is precipitated in plasma. A sublethal dose of the stock solution of sodium oxalate (0-2 ml./100 g) was injected during a period of 5 sec as already described. The needle, still in the vein, was rinsed quickly with 0-1 ml. of normal saline and then a single tracer dose of 47Ca in 0-1 ml. saline was injected at a specified time (10 or 30 sec, 1, 2, 3, 4, 5 or 6 min) after the dose of sodium oxalate. A blood sample was then rapidly taken from the heart. This blood was then left at room temperature for 2 hr to allow complete precipitation of calcium oxalate, and samples of whole blood and plasma were taken for determinations of radioactivity. Lethal dose. In the case of the single injections the lethal dose was considered to be the smallest dose which would cause the death of all rats within 5 min. Disodium EDTA and sodium citrate nearly always caused death within 1 min, or else the animals recovered, but sodium oxalate was very variable in its speed of action and deaths occurred over a range of min. In the chronic injections the lethal dose was considered to be the dose which had been administered up to the time when the rats stopped breathing. The usual course of events was as follows: after a certain time during the chronic infusions, depending on the rate of calcium removal, the rats' breathing became laboured, then erratic and finally stopped; when no further respirations occurred for 30 sec the animal was considered to be dead and the time of the last breath was recorded as the end point-very few animals revived after this stage. Radioactivity determinations were carried out in a scintillation counter with a well-type sodium iodide crystal. All samples were enclosed in a lead sleeve 5 mm thick to absorb the y-rays from 47Sc, the daughter isotope of 47Ca. Packed cell volume of blood samples was determined with the aid of a Hawksley microhaematocrit centrifuge. RESULTS Single injection of disodium ED TA, sodium oxalate and sodium citrate The relative toxicity of disodium EDTA, sodium oxalate and sodium citrate was assessed by first estimating the lethal doses for each when given by the single injection method. The amount of calcium equivalent to this was then calculated and expressed as the 'calcium equivalent' of the lethal dose in mg Ca/100 g rat. The smaller this value the more toxic is the agent and vice versa. Table 1 shows the calcium equivalents of the lethal doses of disodium EDTA, sodium citrate and sodium oxalate.

3 TOXICITY OF SODIUM COMPOUNDS 615 Disodium EDTA is four times more toxic than sodium citrate and nearly ten times more toxic than sodium oxalate. However, the addition of a small amount of calcium oxalate precipitate to the sodium oxalate increases its toxicity. On the other hand, calcium phosphate has no effect. TABLE 1. The lethal doses of disodium EDTA, sodium citrate and sodium oxalate when administered by single intravenous injection Calcium equiv. of lethal dose (mg Ca/100 g rat) Disodium EDTA Sodium citrate Sodium oxalate (CaOX 0-1) Sodium oxalate (CaOX 0.01) (Cas(Po4)2 001) (Ca3(Po4)2 0.01) TABLE 2. The amount of calcium theoretically chelated or removed by disodium EDTA, sodium oxalate and sodium citrate before death occurs, during chronic infusions at various rates Calcium chelated or removed before death (mg/ 100 g) by Rate of Ca chelation or removal (mglmin/100 g) Disodium EDTA Sodium oxalate Sodium citrate _ 5* * Table 2 shows the relation between the theoretical rate of removal of calcium during the chronic infusions (mg/min/100 g) and the total calcium removed by the time the rat had died. These results have been obtained from graphs showing the relation between the -rates of calcium removal or chelation and the time to kill for each agent, plotted as in Payne & Sansom (1963). Few rats were used to assess the toxicity of sodium citrate because it was not very toxic and the injection had to be continued for long periods. The distribution of 47Ca between plasma and whole blood in normal rats and in rats treated with sodium oxalate When a blood sample was taken from a rat shortly after the intravenous injection of a tracer dose of 47Ca, the radioactivity was confined to the

4 616 J. M. PA YNE AND B. F. SANSOM plasma and did not appear to enter the blood cells even after several hours at room temperature. This suggests an alternative method for determining the packed cell volume (P.c.v.). Indeed, calculations based upon the following formula counts/ml. plasma 100 counts/ml. whole blood 100-P.C.V. (%) give very similar results to those determined by the orthodox microhaematocrit method (Table 3). However, when a tracer dose of 47Ca was injected intravenously into a rat after the administration of stock sodium TABiE 3. The relation between the ratio of 47Ca concentration in plasma and whole blood after a single intravenous injection of 47Ca, and the packed cell volume Counts/ml. plasma Calculated Rat P.c.v. (%) counts/ml. whole blood P.c.v. (%)* * * * Where counts/ml. plasma 100 counts/ml. whole blood 100-P. c.v. TABLE 4. The relation between the ratio of 47Ca concentration in plasma and whole blood after a single intravenous injection of 47Ca at intervals after administration of sodium oxalate, and the calculated relative amounts of sodium oxalate remaining unprecipitated as calcium oxalate at these intervals Interval between administration of sodium oxalate and Counts/ml. plasma calcium-47 counts/ml. whole blood Y % (sec) (min) Mean + S.E. Mean + S.E * *67+0* *78+0* *6 2 0*95+0* *4 3 1*16+0* * *2 5 1*61+0*23 7+1*0 6 1* oxalate solution, the calculations did not always hold good. The ratio of the concentration of radioactivity in the plasma to that in the whole blood was not related to the haematocrit in this way, unless the interval between the administration of the sodium oxalate and the tracer was at least 6 min. At all intervals shorter than this the ratio (counts/ml. plasma): (counts/ml. whole blood) was less than 1F72, giving a low value for the haematocrit. The obvious way of accounting for this is to suggest that some radioactivity had been removed from the plasma and spun down with the cells.

5 TOXICITY OF SODIUM COMPOUNDS 617 Table 4 shows the values of the ratio of (plasma radioactivity): (wholeblood radioactivity) at different intervals between the administration of the standard dose of sodium oxalate and the tracer dose of 47Ca, each value being the mean of five determinations. If oxalate takes an appreciable time to precipitate plasma calcium as calcium oxalate, the amount of 47Ca label in the precipitate will depend on the length of time between the injection of oxalate and the isotope. If the interval is brief, then most of the oxalate will still be free at the time the isotope is injected and the resulting precipitate will be radioactive. Conversely, a longer interval will result in a precipitate relatively free from 47Ca. This suggests a method for calculating the time calcium oxalate takes to be precipitated in the blood plasma after the injection of sodium oxalate. The percentage of the tracer precipitated (say Y %) should be proportional to the quantity of free oxalate remaining at the time of the injection, and as the precipitated calcium oxalate is spun down with the blood cells when the blood is centrifuged for the separation of plasma, the ratio of radioactivity in plasma to that in whole blood will be counts/ml. plasma counts/ml. whole blood 100- Y 100-P.C.V. As has been explained above, Y is large if the tracer is injected very shortly after the sodium oxalate and the ratio of the radioactivity in plasma to that in whole blood is low. Conversely, if the tracer is not injected until all the sodium oxalate has been removed as calcium oxalate all the radioactivity will remain in the plasma and the ratio of counts in the plasma to counts in whole blood will be in the same proportion as that predicted by the P.C.V. determination. Table 4 shows the values of this ratio and the values obtained for Y by substituting them in the above equation. DISCUSSION The basic assumption of this work is that the toxicity of disodium EDTA, sodium oxalate and sodium citrate when administered intravenously is due to their ability to chelate or precipitate plasma calcium. Equivalent amounts of these agents should be equally toxic and when injected intravenously should cause death when the concentration of ionic calcium in the blood is reduced below a certain minimum. It is possible to calculate a theoretical lethal dose for each in terms of calciumremoving power. The mean plasma volume is 6-2 ml./100 g rat and the mean plasma calcium concentration is 9-4 mg/100 ml. (Payne & Sansom, 1963), giving a mean total plasma calcium reserve of 0 49 mg Ca/100 g rat. Death should therefore occur before this quantity of calcium is removed.

6 618 J. M. PA YNE AND B. F. SANSOM Experimentally this is found to be true in the case of disodium EDTA, in which the lethal dose by rapid intravenous injection corresponds to the removal of 0 45 mg Ca/100 g rat. Death usually occurs within 15 sec at this dose level and virtually immediately at any higher dose. Lower doses cause considerable respiratory distress with slow recovery. It is therefore concluded that disodium EDTA is a rapid and effective chelating agent when given by rapid intravenous injection. However, when injected slowly its toxicity is reduced. This might be expected, because the longer time allows sources of calcium other than those contained in the plasma alone to be called into use. Even so, the amount of calcium removed before death remains nearly constant at about 1.0 mg over a range of doses from 0-48 to 0-15 mg/100 g/min. This is twice the amount removed by the acute lethal dose. It only increases when the dose rate is further reduced. The acute lethal doses of sodium citrate and sodium oxalate are 4 and nearly 10 times respectively that for disodium EDTA. There are three possible reasons for this: (1) the chelates formed are less stable; (2) the citrate or oxalate ions may be metabolized and rendered harmless or (3) there may be a time lag between the injection of the agent and the removal of the plasma calcium. This would apply particularly in the case of oxalate, where the precipitation of calcium oxalate may be delayed because of a supersaturation phenomenon. The stability of the calcium citrate complex is less than that of calcium EDTA, the logarithms of the stability constants being 4 90 and respectively. Moreover, it is well known that citrate is metabolized in the Krebs cycle. These are probably the main reasons for the very low toxicity of sodium citrate when injected slowly; the chelate must be readily broken down and the citrate ion metabolized. It is interesting that in the acute tests sodium citrate was nearly as toxic as disodium EDTA and certainly more toxic than sodium oxalate. However, the toxicity falls rapidly as the dose rate is lowered. It is therefore concluded that sodium citrate has a fairly high initial chelating power, but that the stability of the chelate formed is low. The free citrate ions thus formed appear to be rapidly detoxicated by the body tissues. The low acute toxicity of oxalate is partly attributable to a delay in the precipitation of calcium oxalate or to a supersaturation phenomenon which gives the animal time to mobilize more calcium than is available in the blood plasma alone. The experiments with sodium oxalate containing a suspension of preformed calcium oxalate seem to confirm this. Thus the presence of very small amounts of calcium oxalate in the sodium oxalate solution reduces the minimum lethal dose of sodium oxalate from the equivalent of 4-2 to 1-8 mg Ca/l00 g. Increasing the amount of

7 TOXICITY OF SODIUM COMPOUNDS 619 calcium oxalate tenfold only reduces it a little further, to 1-2 mg Ca/100 g. On the other hand, equivalent amounts of calcium phosphate are completely without effect, indicating that the action of the calcium oxalate is specific rather than due to the gross physical action of solid particles in the injection fluid. The results of the experiments in which 47Ca was injected intravenously at varying intervals after sodium oxalate give further confirmation of the hypothesis that oxalate is less toxic than disodium EDTA because of the time lag in precipitation. It is possible to calculate the length of time involved. Thus it appears that free oxalate ions are still precipitating calcium up to 6 min after the injection. During the period of delay some mobilization of calcium from reserves outside the blood will almost certainly occur. Indeed, in the experiments with disodium EDTA the available calcium was doubled in 6 min. It might therefore be predicted that the acute lethal dose of sodium oxalate would be only twice that of disodium EDTA instead of nearly tenfold, as it actually is. It therefore seems probable that some additional process other than precipitation removes oxalate ions from plasma. If this process is faster the higher the concentration of oxalate, then we might explain the other curious feature of sodium oxalate toxicity, that it increases with decreasing dose rate. Such a process might depend on renal excretion. The faster the dose rate the higher the plasma concentration of oxalate and the larger the proportion of the dose excreted by the kidney before the oxalate is precipitated as calcium oxalate. At lower dose rates a larger proportion of the dose may be involved in precipitating calcium as calcium oxalate. The conditions under which an acute dose of sodium oxalate is most toxic is in the presence of preformed calcium oxalate, which speeds up the process of precipitation and so prevents the kidney excreting such a high proportion of the dose. It may be concluded that disodium EDTA is a completely effective agent for the chelation of plasma calcium in vivo. Sodium oxalate is fairly effective if administered at low dose rates, but less effective when administered rapidly. Sodium citrate is only effective if administered rapidly and becomes almost non-toxic at low dose rates. Evidence will be presented in another paper of the routes of excretion of disodium EDTA and sodium oxalate. SUMMARY 1. The relative efficacy of disodium EDTA, sodium oxalate and sodium citrate for inducing experimental hypocalcaemia has been determined by comparing their toxicity to rats when administered intravenously. 2. Disodium EDTA is highly toxic and appears to chelate plasma calcium rapidly and effectively.

8 620 J. M. PAYNE AND B. F. SANSOM 3. Sodium oxalate is much less toxic, but its toxicity is specifically increased by the presence of preformed calcium oxalate crystals. 4. Radioisotope experiments indicate that sodium oxalate requires about 6 min to precipitate its equivalent of calcium as calcium oxalate completely. 5. Sodium citrate is highly toxic when given by rapid intravenous injection but its toxicity rapidly declines as the rate of administration is reduced. REFERENCES PAYNE, J. M. & SANSOM, B. F. (1963). The susceptibility experimental hypocalcaemia. J. Physiol. 168, of various groups of rats to PAYNE, J. M., SANsom, B. F. & MANSTON, R. (1963). The responses of cows to experimentally induced hypocalcaemia. 1. Acute experimental hypocalcaemia. Vet. Rec. 75, 588.