for Research on Animal Diseases, Compton, Newbury, Berkshire (Received 23 May 1967)

Transcription

1 J. Phy8iol. (1967), 193, pp With 4 text-figures Printed in Great Britain THE EXPERIMENTAL INDUCTION OF HYPOPHOSPHATAEMIA IN GOATS USING ANION EXCHANGE COLUMNS By H. W. SYMONDS AND R. J. TREACHER From the Department of Functional Pathology, A.R.C. Institute for Research on Animal Diseases, Compton, Newbury, Berkshire (Received 23 May 1967) SUMMARY 1. Inorganic phosphate was selectively removed from the circulating blood of five anaesthetized goats by means of columns of anion-exchange resin. Concentrations of other ions were unaffected by the column. 2. Rates of inorganic phosphate extraction in excess of 5 m-moles/hr caused a fall in plasma inorganic phosphate of up to 50 % in 30 min. 3. In four of the goats the calculated inorganic phosphate mobilization rate rose sharply to a maximum between 5-8 and 12-7 m-moles/hr and then fell progressively as more phosphate was removed. 4. During the course of the experiments a fall in blood pressure occurred, accompanied by a rise in blood glucose. The reasons for this are discussed. INTRODUCTION In investigations into the aetiology of milk fever in the cow Payne (1964) used the removal of plasma calcium by ethylenediaminetetra-acetate (EDTA) infusion to estimate the cow's calcium mobilization rate. The estimation of the phosphate mobilization rate may also provide useful information, but there has been no known method for selectively removing phosphate from circulating blood. This paper describes a new technique for doing this, employing a column of anion exchange resin equilibrated so as to remove phosphate from the blood in vivo while leaving other ions unaffected. The technique was applied to goats and the results obtained have been used to calculate the rate at which the plasma phosphate can be replenished. METHODS The apparatus is shown diagrammatically in Fig. 1. The column (42 x 7-8 cm) was packed with De-acidite FF anion exchange resin (-14 to + 52 mesh 3.5 % cross-linkage [Permutit Co. Ltd.]) prepared as described by Kumar & MacIntyre (1963) for cationic resins. The resin had been previously washed several times alternately with 2N-NaOH and 2N-HC1. The

2 620 H. W. SYMONDS AND R. J. TREACHER resin in the chloride form was then equilibrated for several hours with several changes of an equilibrating fluid. This contained the major electrolytes of blood plasma except phosphate and had been bubbled for several hours with 5 % carbon dioxide in oxygen. The detailed composition of this fluid was as follows (m.equiv/l.): Na+ 136, K+ 3-6, Ca , Mg , C1-116, S , HCO3-26-7, and glucose 3 0 m-moles/l. After bubbling with 5% C02 in 02 lactic acid was added to ph 7-3. Sample point. _ Stainless- :-.De-bubbler - steel mesh _. Anion- ::Blood to exchange jugular vein column -... texthermostated By-pass - water-bath 390 C tube i...seheating coil Sample Peristaltic pumps Fig. 1. Diagram of apparatus for phosphate removal. Blood from jugular vein Hepari n The resin was packed into the column and equilibrating fluid pumped through at 40 ml./ min for 15 hr, until the chloride concentration in the fluid leaving the collumn equalled that entering the column. Tmmediately before use the whole apparatus was washed through with fresh equilibrating fluid containing 5 g Dextran (average mol. wt. 115,000, Glaxo Laboratories Ltd.)/100 ml. A column prepared in this way removed phosphate but no other major ion from the blood passing through it. After use the total amount of phosphate absorbed by the column could be determined by washing the resin with 2N-HCI and estimating the amount of phosphate in the washings. The plasma volume of each goat was estimated by the Evans Blue intravenous dye dilution method (Noble & Gregerson, 1946). Experimental technique. Adult female goats aged 2-4 years and weighing kg were used. An initial jugular blood sample was collected, and anaesthesia induced with methohexitone sodium and maintained with a cyclopropane/oxygen mixture using a circle absorber system. With the animal in dorsal recumbency about 3 in. (7-6 cm) of the left carotid artery and jugular vein were exposed and carefully separated from adjacent connective tissue. Care was taken to avoid damage to the vagus nerve when handling the carotid. A tracheotomy was then performed and a glass cannula inserted to provide a more open airway. This was important because, as will be seen later, respiratory embarrassment in conjunction with high blood heparin levels may cause lung haemorrhages. Experience had shown that less lung damage occurred when the goat was in lateral recumbency, so before injecting heparin the goat was turned on to its right side. Ten thousand units of heparin were then given intravenously in preparation for cannulation of carotid and jugular vessels and a second

3 HYPOPHOSPHATAEMIA IN GOATS 621 jugular blood sample was taken. Mean arterial blood pressure was registered by a mercury manometer attached via a glass Y-piece into the carotid artery. This system allowed normal blood flow to the head. The exposed jugular vein was clamped, transected and the apparatus connected to it via glass cannulae as shown in Fig. 1. Blood was pumped through the apparatus by a peristaltic pump at a measured rate (usually about 200 ml./min) and returned to the jugular vein. Additional heparin was added to the circulating blood at a rate of 2000 units/min using a second pump. For the first 30 min following completion of the preparatory procedure blood flowed through the apparatus but by-passed the column. Three blood samples were collected at 10 min intervals during this period. Immediately after the last of these samples was taken, the blood flow was diverted through the column. Starting 4 min after this, samples of blood entering the column were taken at 5 min intervals. In addition a sample of blood leaving the column was taken 2 min after each sample taken entering the column. The 2 min interval allowed for the time taken for blood to flow through the column. Thirty minutes after the blood flow through the column began the flow was rediverted via the by-pass tube. Blood samples were collected every 5 min during the first quarter of an hour thereafter and subsequently at 10 min intervals. At the end of the experiment the animal was killed while still anaesthetized by bleeding from the carotid artery. Urine was collected from an in-dwelling balloon catheter placed in the bladder through a mid line laparotomy incision. The goat's head was allowed to hang downwards and all saliva was collected into a small bowl. Two goats were subjected to a full control experiment using a column fully equilibrated with a phosphate concentration equal to that of the goat's plasma before experiment. In addition a series of control experiments were carried out to establish to what extent the individual techniques making up the procedure contributed to the biochemical and clinical effects noted in both the full control and the removal experiment. The techniques examined were (i) the effect of anaesthesia and the position of the goat. (ii) The effect of the surgical interference. In this experiment a 6 in. (15.2 cm) length of silicone rubber tubing represented the extraction apparatus in the transected jugular. (iii) The effect of the dilution of the systemic blood with equilibrating fluid. The resin column was replaced by a vessel of volume equivalent to that of the void volume of the resin column and the goat's blood continuously recycled through it. (iv) The possibility of toxic products leaching off the column. Chemical analy8i8. Plasma inorganic phosphate (Pi) was determined by the autoanalyser modification of the method of Fiske & Subbarow (1925). Total Pi in plasma, whole blood, urine and saliva was estimated by autoanalysis of aliquots wet ashed with concentrated nitric and sulphuric acids. For total Ca in urine and saliva the autoanalyser method of Skeggs & Stevens (1960) was used on wet ashed samples. Plasma sodium and potassium were determined on a Unicam SP 900 flame photometer, plasma Ca and Mg by a modification of the method of Bowden & Patson (1963) using an EEL titrator, plasma Cl by titration with mercuric nitrate and protein by measuring the absorbency of a 1: 200 dilution of plasma at 275 mg on a Unicam SP 500 spectrophotometer. Post mortem examination. A routine post mortem examination of the lungs was carried out to determine the effects of large doses of heparin. MATHEMATICAL INTERPRETATION OF RESULTS The purpose of these experiments was to remove inorganic phosphate from the blood plasma of goats at a rate faster than it could be replaced, thus causing the plasma phosphate concentration to fall. The rate of phosphate removal is controlled by the rate of blood flow through the

4 622 H. W. SYMONDS AND R. J. TREACHER column, and can be adjusted to some extent by altering the speed of the pump. The maximum blood flow is, of course, limited by the volume of venous return from the head via the jugular vein. If the flow rate through the column and the difference between the phosphate concentration in the blood entering and leaving the column is known then the rate of removal of phosphate can be calculated for each 5 min sampling interval using the plasma inorganic phosphate concentration (mm) in the blood samples, as follows: B1 = Blood entering the column at start time T. B2 = Blood entering the column at end time T + 5 min. A1 = Blood leaving the column at start time T+ 2 min. A2 = Blood leaving the column at end time T+ 7 min. F = Plasma flow rate through the column (ml./min). V = Plasma volume of animal (ml.). Then the rate of removal of inorganic phosphate by the column (B1- A1) + (B2- A2) x F x 60 l.moles/hr, 2 x 1000 and the rate of fall in total plasma inorganic phosphate (B1-B2) X V x 60m.moles/hr x 5 Then the rate of removal of inorganic phosphate by the column minus the rate of fall in the total plasma inorganic phosphate gives the rate at which inorganic phosphate enters the vascular system in m-moles/hr. This figure has been termed the 'mobilization rate' but this must be distinguished from the rate of movement of phosphate into the total immediately available 'pool' of phosphate, which would include that of the extracellular fluid and a small proportion of the skeleton in addition to that in the plasma. The volume of this 'pool' would have to be determined or assumed before mobilization into this pool could be calculated from the present experimental data. RESULTS The plasma inorganic phosphate concentrations, entering and leaving the column, in goat E are shown graphically in Fig. 2. The calculated mobilization rates for the five goats from which phosphate was removed are presented in Table 1 and the results from goat E are shown in Fig. 3. In four of the experiments the mobilization rate rose sharply to a maximum within the first 5 min of the column beginning to operate and began

5 HYPOPHOSPHATAEMIA IN GOATS 623 to fall after min as the phosphate removal proceeded, in spite of the fact that the plasma inorganic phosphate level was falling. The rate of phosphate removal from the fifth goat (B) was limited by the rate of venous return from the head and the rate of fall of plasma phosphate TABLE 1. Phosphorus mobilization rates in goats Blood flow Total P Plasma through amount mobilization Weight vol. column Pi removed ratet Goat no. (kg) (ml.) (ml./min) (m-moles) (m-moles/hr) A B* C D E F (control) G (control) * Extraction for 1 hr. t Figures represent the animals' maximum Pi mobilizing rate, except in the case of goat B and the two controls *0 E I -06 _ 04_ ~~~~ I 'A I I I I m a Minutes Fig. 2. Plasma inorganic phosphate concentrations during phosphate removal experiment (goat E). 0 Jugular blood. 0 Blood leaving column. Dashed lines show the time during which the column was in the circuit. concentration was very low. It is probable that the removal rate was insufficient to cause the maximum possible mobilization rate to be reached. In all five experiments the mobilization rate remained low during the recovery phase after the column had been removed from the circuit. The plasma inorganic phosphate had returned to the pre-experimental concentration within 80 min of the column being removed. In the full control experiments the column did not remove phosphate immediately after it was placed in the circuit, but the concentration of inorganic phosphate in the blood leaving the column slowly fell to 75 % of the level of that entering the column over the next 30 min. The haematocrit value fell by up to 10 % immediately after anaesthesia

6 624 H. W. SYMONDS AND R. J. TREACHER in all five phosphate removal experiments. Subsequently there was little further variation in the value. A similar change occurred in control experiment (i) I/v\s I I~~~~~~~I ~ 'I Minutes Fig. 3. Net movement ofphosphate into plasma in mm/hr during a phosphate removal experiment on goat E. Dashed lines show the time during which the column was in the circuit. Whole blood total phosphate and plasma total phosphate concentrations were measured in only two goats and were observed to fall during phosphate removal. This fall could be accounted for by the reduction in the plasma inorganic phosphate concentration and haematocrit values. Plasma organic phosphate concentration and red blood cell phosphate remained constant throughout. There was no significant change in the plasma concentrations of magnesium, sodium, chloride and potassium, although potassium concentrations were sometimes erratic. A fall in the plasma calcium from 4-6 to 4*25 m-equiv/l. occurred. A similar fall occurred in the full control experiment but not in control experiments where no resin column was used. The fall observed in plasma protein concentration during phosphate extraction could be almost totally accounted for by dilution of the plasma with equilibrating fluid present in the apparatus and the loss of protein due to blood sampling. Changes in blood glucose concentration varied. In three experiments there was a rise of from about 2-78 to 8-34 m-moles/l. and in one a rise from 8-9 to m-moles/l., while a slight fall was observed in one goat. The fluctuations did not coincide with the period of operation of the column. A similar variation occurred in all the control experiments.

7 HYPOPHOSPHATAEMIA IN GOATS 625 Apart from glucose virtually identical qualitative blood pictures were thus obtained in all of the five animals used. A fall in blood pressure to below 70 mm Hg occurred in four experiments. In the fifth experiment the pressure did not fall below 120 mm Hg. Similar falls occurred in all the control experiments where a column was used or where a large volume of equilibrating fluid was infused into the animal. The volume and phosphate concentrations of the saliva in goat E are shown in Fig. 4. Salivary secretion fell progressively during the course of the experiments but this trend was established from the beginning of 250 r, * ~ r\ 4.0 E 100 I2.0 E 50 o Minutes Fig. 4. Volume and phosphate concentration of saliva produced over 2j hr by goat'e during a phosphate removal experiment. * Voluime. 0 Phosphate secretion rate. Dashed lines show the time during which the column was in the circuit. anaesthesia and did not depend on the presence of the column. Phosphate concentration in the saliva varied very little. In the full control experiments where very little phosphate was removed the fall in saliva volume was rather more rapid and there was no change in the phosphate concentration. The calcium and phosphate excretion in the urine was very low (less than m-equiv/15 min calcium and less than m-moles/15 min phosphate). Post mortem examination. Post mortem examinations revealed pulmonary congestion of varying severity. The changes were most pronounced in the diaphragmatic and cardiac lobes of the right lung and in the aortic and oesophageal groove region of both lungs. 40 Physiol. I93

8 626 H. W. SYMONDS AND R. J. TREACHER Haematological examination. Microscopical examination of the blood leaving the resin column showed that the red cells were normal in number and shape but there was a reduction in the number of leucocytes. DISCUSSION The technique described in this paper has been shown to be a selective and efficient method of removing inorganic phosphate from the circulation. It is also relatively trouble free in that the blood does not clot in the apparatus provided sufficient heparin is used. No haemolysis has been observed. The reason for the substantial fall in blood pressure during most of our experiments remains unknown. A control experiment suggests that it is a feature of anaesthesia but that other factors may also be involved. The possibility that toxic amines released from the column resin were responsible is ruled out because the fall sometimes started before the column was placed in the circuit. Furthermore, a similar fall in blood pressure occurred after the infusion of 400 ml. of fresh equilibrating fluid. The possibility that this is a response to pyrogens in the infusion fluid is being investigated. Greeberg, Visscher, Petersen & Boyd (1942) found severe pulmonary congestion developed in goats kept upon their backs for several hours and suggested that this was due to the position of the heart restricting the venous return from the lungs. For this reason we put our goats into lateral recumbency immediately surgery was complete and before administering any heparin. However, this was not sufficient to prevent a steadily increasing state of anoxia. In general the concentrations of the various ions other than phosphate in blood and saliva were similar in the control goats and in the animals from which phosphate was removed. The rise in blood glucose occurred in all the animals including the controls and appears to be associated with anaesthesia and is not related to surgical interference. The 10 % fall in plasma calcium occurred in all goats where a column was used, whether to remove phosphate or not. It therefore appears that the alterations observed in the plasma phosphate concentrations are in response to the removal of phosphate and that our observations on phosphate mobilization are valid. The control experiments show that plasma inorganic phosphate concentrations change slowly over long periods in the preparation but are not influenced by the surgical manipulations themselves. The maximum mobilization rates for phosphate ranged from 5x8 to 12*7 m-moles/hr. The form of the phosphate mobilization curve is interesting, since the maximum mobilization level is reached extremely rapidly (usually within the first 5 min of phosphate removal) and when the fall in

9 HYPOPHOSPHATAEMIA IN GOATS 627 plasma level is barely detectable. In four of the five goats after min the mobilization rate fell sharply, suggesting that an initial source of phosphate had become depleted. After the column had been removed from the circuit, the recovery of the plasma phosphate concentration was slow, but in all animals it returned to its pre-experimental level within 80 min. It appears on this evidence that there is some mechanism regulating the plasma inorganic phosphate concentration which is sensitive to small falls in concentration and that at least one source of immediately available phosphate is soon exhausted. If such a mechanism exists it cannot be very effective because it is well known that the plasma inorganic phosphate concentration normally undergoes large spontaneous variations. Furthermore, it appears from the control experiments, where all the saliva produced was collected, that the goat can lose large amounts of phosphate in the saliva (equal to that removed by the column in the same period) without experiencing a fall in the plasma inorganic phosphate concentration. Possible sources from which phosphate might be mobilized to meet the drain imposed by the ion exchange procedure are saliva, organic plasma phosphate, bone, intracellular phosphate (including erythrocyte phosphate), intestinal phosphate and increased reabsorption of phosphate by the kidney tubules. The first two sources can be ruled out as there was no significant change in the amount of phosphate secreted in the saliva compared with control experiments, nor was. there any fall in plasma organic phosphate concentration during Pi removal. Phosphate excretion in the urine was so low that any fall in excretion would be too small to detect using the analytical and sampling methods employed and is therefore unlikely to be of importance in mineral metabolism. Bone does not seem to be a likely source because of the rapidity with which the mobilization occurs and the fact that no concomitant increase in plasma or urine calcium was detected. Indeed a decrease in concentration normally occurred. The results did not indicate that the concentration of phosphate in the erythrocytes was lowered during plasma or inorganic phosphate removal. No measurements were made of changes in cellular phosphate of other tissues but this must be considered a likely source of considerable quantities of available phosphate. It has been shown that phosphate can be transferred from the gut to the circulation in less than 5 min in the dog (Weissberger & Nasset, 1942) although the presence of an active transport mechanism has yet to be finely established. Scarisbrick & Ewer (1951) were able to raise the plasma inorganic phosphate concentration in sheep from 2-26 to 6 45 m-moles/l. in only 10 min, by placing a high concentration of inorganic phosphate directly into the rumen. 40-2

10 628 H. W. SYMONDS AND R. J. TREACHER No measurements of the phosphate content of either the intestinal lumen or the walls of the alimentary tract were made during these experiments, but it seems quite likely that this may provide a considerable pool of available Pi when plasma concentration is reduced. We cannot explain the 5-10 % fall in plasma calcium values observed in all experiments where a column was used (whether control or not). Calcium was not extracted by the column, none of the other electrolytes was affected, and there was no evidence that protein was extracted by the column. The fate of the calcium lost from the plasma and the reason it was not replaced are unknown. Mild hyperglycaemia is an acknowledged feature of cyclopropane anaesthesia (Westhues & Fritsch, 1965) and in view of the pulmonary congestion found post mortem the very large rises observed in some of our experiments can probably be attributed to a progressively increasing degree of anoxia. The behaviour of the column during control experiments, where the resin had previously been equilibrated with inorganic phosphate to the level of that in the goat's plasma before the experiment began, is puzzling. The continually increasing capacity of the column to capture phosphate might be due to the absorption on to it of some ion from the plasma which itself could bind phosphate, such as calcium or protein. However, since neither calcium nor protein was removed from plasma passing through the column, the cause remains unknown. The experiments demonstrate that in the goat in response to even a small depletion of the plasma inorganic phosphate, there is a net movement of inorganic phosphate into the plasma (and thus first into the extracellular fluid) most probably from intestinal and intracellular sources. These facts indicate the existence of some mechanism controlling plasma inorganic phosphate levels, either directly or indirectly. We wish to thank Mrs J. Barlow and Mr B. Turfrey for technical assistance. REFERENCES BOWDEN, C. H. & PATSON, V. J. (1963). Microdetermination of calcium and magnesium in biological material. J. clin. Path. 16, FisKE, C. H. & SuBEBAROW, Y. (1925). As modified in Autoanaly8er Method File. New York: Technicon Instrument Corporation. GREEBERG, A. J., VIsscirER, M. B., PETERSEN, W. E. & BOYD, W. A. (1942). The cause of death in ruminants held on their backs. J. Am. vet. med. A , KUMAR, M. A. & MACINTYRE, I. (1963). Use of ion exchange resins to alter the concentrations of individual inorganic ions in blood in vivo. Nature, Lond. 200, 181. NOBLE, R. P. & GREGERSON, M. T. (1946). Blood volume in clinical shock. Mixing time and disappearance rates of T 1824 in normal subjects in patients in shock. J. clin. Invest. 25, PAYNE, J. M. (1964). The responses of cows to experimentally induced hypocalcaemia. Vet. Rec. 76,

11 HYPOPHOSPHATAEMIA IN GOATS 629 ScAsisBRIcK, R. & EwER, T. K. (1951). Absorption of inorganic phosphate from the rumen of sheep. Biochem. J. 49, 79. SKEGGS, L. & STEVENS, D. L. (1960). Calcium II. Autoanalyser Method File. New York: Technicon Instrument Corporation. WEISSBERGER, L. H. & NASSET, E. S. (1942). Phosphorus absorption in the anaesthetised dog as determined with radioactive isotopes. Am. J. Phy8iol. 138, WESTHUES, M. & FRITScH, R. (1965). Animal Anae"the8ia, vol. ii, p. 69. Edinburgh: Oliver and Bovd.