PHOSPHATE HOMEOSTASIS IN ADULT SHEEP. By G. H. MCINTOSH and F. M. ToMAs. From the CSIRO Division of Human Nutrition, Adelaide.

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1 Quarterly Journal of Experimental Physiology (1978) 63, THE INFLUENCE OF PARATHYROIDECTOMY UPON CALCIUM AND PHOSPHATE HOMEOSTASIS IN ADULT SHEEP. By G. H. MCINTOSH and F. M. ToMAs. From the CSIRO Division of Human Nutrition, Adelaide. (Receivedfor publication 1st June 1977) The absorption and secretion of Ca and P04 were measured with the use of radioactive isopes in 4 adult sheep before and after parathyroidecmy. Secretion of Ca and P04 in the smach and intestines was measured separately. Parathyroidecmy resulted in a negative balance for Ca and P04 and an accompanying fall in plasma Ca and P04 concentrations. In the case of P04 this response was attributed primarily a reduced intestinal absorption, but for Ca increases in urinary exretion and intestinal secretion also contributed significantly. Secretion of P04 the smach was reduced but endogenous P04 excretion in the faeces was unchanged which indicated a reduced reabsorption of secreted P04 by the intestines. Mayer, Ramberg and Kronfeld [1967,1970] have observed that parathyroidecmy (PTX) of cattle increased faecal excretion of Ca and P, but were unable determine whether this was due reduced absorption ofthese elements or increased secretion. Although there now appears be some doubt as whether endogenous faecal Ca is increased [Kronfeld, Mayer and Ramberg, 1976] the increase in faecal P after PTX is generally accepted. One of the major vehicles of P04 secretion in the gastrointestinal tract of ruminants is the saliva [Kay, 1966] and in acute studies Tomas [1974] and Clark, French, Beale, Cross and Butz-Olsen [1975] have shown that salivary P04 is increased following parathyroid extract administration. This finding suggests that it is reduced absorption of P after PIX rather than increased secretion which is responsible for increased faecal P. The relevance ofthese questions in establishing the role ofparathyroid hormone in Ca and P04 homeostasis prompted this study. Radioisopes were used in order measure the exchange of Ca and P04 across the gut wall in normal and parathyroidecmized sheep. METHODS Animal preparation and diet Four adult Merino wethers of kg body weight were each surgically prepared with a moulded T-shaped cannula inserted in the duodenum adjacent the pylorus. The sheep were held in metabolism cages and fed 750 g.d-& of pelleted lucerne hay delivered aumatically in equal hourly aliquots. This ration, which provided 206 mmol Ca. d-1 and 47 8 mmol P04. d- 1 throughout the experiment, was also fed the sheep for 6 weeks beforehand. Ad libitum water consumption was measured and the water sampled for mineral analyses. Intake of Ca via drinking water varied from mmol.d-1. Urine was funneled directly in polyethylene bottles containing 50 ml 12 N HC1 by a moulded collecr which was attached each sheep's belly around the prepuce. For the injection of radioisopes and blood sampling an indwelling cannula was established in each jugular vein. 119

2 120 McInsh and Tomas Experimental procol and procedures There were two experimental periods of 13 days duration, one before and one after parathyroidecmy. Each period commenced with an injection of 1 mci 32P (sodium orthophosphate) intravenously each animal. Total daily collections of urine and faeces were taken over the next 3 days and blood (10 ml) and duodenal digesta (15-25 ml) samples removed at 6, 12, 24, 48, and 72h following injection in order assess the times of appearance of peak radioactivity. On the sixth day, an intravenous injection of 50,uCi 45Ca was given and daily collection of urine and faeces and sampling of blood and duodenal fluid continued for 7 days thereafter. Blood samples were centrifuged immediately and the plasma sred at - 5 C. Duodenal digesta and urine samples were sred at 0-4 C. Aliquots of daily faecal output were dried for 24 h in an oven at 106 C and then sred separately in plastic bags. Parathyroidecmy Upon completion of the first experimental period the sheep were parathyroidecmized by removing the cranial half of each thyroid lobe containing the internal parathyroids, and the external parathyroids [Care, Keynes and Duncan, 1966; Calislar and St. Clair, 1974]. There was little no melanin pigment present in their capsules so that identification of parathyroid tissue was confirmed hislogically. A recovery period of 6 days was allowed before the second experimental period began. All feed intakes returned normal within 24 h of the surgery. At the end of the experiment the sheep were killed and the remaining thyroid tissue removed and examined hislogically. Analytical techniques Finely ground dried faeces and duodenal digesta samples were digested in a nitric-perchloric acid mixture and made up volume. Plasma samples were added 10% trichloro-acetic acid, centrifuged and the supernatant retained. Aliquots of these prepared samples and of urine were placed in counting vials with 10 ml of luene-trin X- 100 scintillar and the 32p and 45Ca radioactivity counted using a Packard Tricarb liquid scintillation spectrometer (Model No. 3001). The samples were analysed for calcium by amic absorption spectroscopy (Techtron AA4) and for phosphate by au-analysis (Technicon Instruments Corporation, Ardsley, New York). Computations Specific activities for Ca and P04 in plasma, duodenal digesta and faeces were calculated daily in similar manner that adopted by Mayer et al. [1967] for Ca and the values plotted on semilogarithmic paper. The data showed a consistent lag of 9 h for peak 32p activity in duodenal digesta and 36 h for 32p and 45Ca activities in faeces relative those in the plasma. The time bases of the curves were offset so that the peaks of radioactivity in the digesta, faeces and blood were synchronized. Daily quantities of endogenous Ca and P04 secretion were calculated from the data obtained in the last 7 d of each period using the formulae: Amount from endogen- Total amount Specific Activity in faeces ous sources in faeces in faeces XSpecific Activity in plasma Amount from endogenous Total amount ( Specific Activity in plasma sources in duodenal digesta = in diet Specific Activity in duodenum 1 The assumptions on which these formulae rely have been discussed by Bronner [1964]. The endogenous secretion the intestines was calculated as the difference between the amounts entering in the digesta and leaving in the faeces. Balances for Ca and P04 were calculated on a daily basis from the amounts in feed, water, urine and faeces.

3 Calcium and phosphate in parathyroidecmized sheep 121 RESULTS Efficacy ofparathyroidecmy procedure Hislogical examination of the thyroid tissue removed post mortem showed that two sheep (Nos. 554 and 525) had been incompletely parathyroidecmized. In each case, the thyroid lobe on the right hand side contained some internal parathyroid tissue, which was estimated be about 10-20% of the original tal parathyroid mass. In these sheep the parathyroid tissue was more caudal in the right than in the left lobe of the thyroid. The remaining thyroid tissue appeared be functionally normal. Plasma Ca and P04 levels There was a sustained fall in plasma Ca and inorganic P04 levels following parathyroidecmy (Table I). The fall was more pronounced for P04 than for Ca (O53 mmolplh vs 009 mmol 1-'). TABLE I. The effect ofparathyroidecmy upon plasma Ca and inorganic PO4 concentrations (mmol. -1) (Each pair of values is the mean and standard error of 7 samples.) Calcium Phosphate Sheep No. 525* * * * 512 Control P *00 +0*028 ±0-045 ±0-017e ± After parathyroidecmy ± ± { ± {071 *Incomplete parathyroidecmy (see text) Endogenous secretion The effects of parathyroidecmy upon the endogenous secretion of Ca and P04 are shown in Table II. Ca secretion in the smach was unaltered, but an increased amount entered the intestines, resulting in an increased faecal excretion of endogenous Ca. Apart from sheep 525, parathyroidecmy led a reduction in P04 secretion the smach. This was especially marked in sheep 512 from which all parathyroid tissue was removed and was associated with the greatest fall in plasma P04 level. The amount of P04 of endogenous origin in the faeces was considerably less than that secreted the smach and was relatively unaffected by parathyroidecmy. Thus, the calculated net reabsorption of secreted P04 by the intestines was substantially reduced after parathyroidecmy (Table II). Absorption, excretion and balances of Ca and P04 The excretion, balances and calculated 'true' absorption of Ca and P04 before and after parathyroidecmy are shown in Table III. Except for faecal Ca in sheep 549, faecal excretion of Ca and P04 was increased following parathyroidecmy, and in large part accounted for the substantial net negative

4 122 McInsh and Tomas balance of these minerals that developed. Urinary P04 excretion remained negligible throughout the experiment but urinary Ca was increased by parathyroidecmy, particularly in sheep 512 and 549. The absorption of dietary Ca and P04 TABLE II. The effect ofparathyroidecmy upon endogenous Ca and PO4 secretion the smach and excretion in the faeces (mmol. d-1) Sheep Calcium Phosphate No. smach intestine in faeces smach intestine in faeces Control & V * V Mean Post PTX 525* * Mean PTX COntrOl *Incomplete PTX TABLE III. The effect of parathyroidecmy (PTX) on the absorption, excretion and balance of Ca and P04 (mmol. d). Calcium Phosphate Sheep in in in in No. urine faeces balancet absorbedt urine faeces balancet absorbedt neglig- Control S ible ,, ,, ,, P3 Mean neglig- Post PTX 525* ible C4 10.1,, 64* * ,, ,, Mean *InCOmplete PTX. tfodder provided 206 mmol Ca.d-1 and 47T8 mmol P04.d1 Drinking water provided mmol Ca/d tabsorbed Ca or P04 = Intake-(Total amount in faeces-amount in faeces of endogenous origin). was markedly reduced by parathyroidecmy except for sheep 549 in which Ca absorption was maintained but urinary Ca excretion increased nearly 5-fold. No explanation can be found for the anomalous negative Ca absorption shown for sheep 554.

5 Calcium and phosphate in parathyroidecmized sheep DISCUSSION The results show that a principal effect of parathyroidecmy (PTX) in adult sheep is a reduction in absorption of both Ca and P04 from the intestines and this effect largely accounts for the ensuing negative balance for both minerals. The decreased absorption may also contribute the lowered plasma Ca and P04 concentrations following parathyroidecmy, although the possibility of a decreased release of minerals from the bone due lack of PTH could also have contributed [Kronfeld, et al. 1976]. In relation homeostasis, other significant changes following PTX were the reduction in secretion of P04 the smach and the increase in secretion of Ca the intestines. The former response is consistent with the observed reduction in plasma P04 levels [Tomas, Moir and Somers, 1967] which in turn can be accounted for by the reduced absorption of P04. However, the alternative possibility that parathyroidecmy removed a direct stimulary effect of PTH on P04 secretion by the parotid salivary glands [Tomas, 1974; Clark et al. 1975] cannot be excluded and warrants further investigation. The observed reduction in Ca absorption by the intestines following parathyroidecmy is in agreement with the findings of Mayer et al. [1967] with cattle and shows that PTH plays an important role in facilitating Ca absorption in ruminants, even when 'chey receive a diet with a high Ca content. In these respects it seems that ruminants differ from the rat in which studies in vivo have shown PTX have a limited effect of Ca absoprtion, discernible only at low Ca intakes [Shah and Draper, 1966; Clark and Rivera-Cordero, 1973; Kemm, 1976]. However, there was a large variation in the Ca absorption response by the sheep (Table III) which may have arisen from post-operative effects. The time allowed for recovery may have been insufficient for complete adaptation parathyroidecmy and could explain, at least in part, the different responses Ca absorption by the animals. Certainly the responses were not consistent with the degree of parathyroid extirpation. The increase in net excretion of endogenous Ca via the intestines is an unexpected finding in view ofthe reduction in both plasma Ca levels and Ca absorption. Kronfeld et al. [1976] also observed an increase (20%) in endogenous Ca secretion in the intestines of cows following parathyroidecmy, although in the light ofsome oftheir earlier data they did not consider it a significant finding. Although the determination of endogenous faecal Ca secretion is sensitive error because of its size relative tal faecal Ca, no systematic error leading the observed increase could be found, and the effect is considered be real; the explanation for the increased secretion remains be clarified. It has been established for both rats and cattle that the removal of Ca from bone is inversely related the amount ofca absorbed. Presumably the mechanism controlling this response can function independently of PTH, since it was shown also occur in parathyroidecmized animals, albeit the rate of exchange was slowed [Sammon, Stacey and Bronner, 1970; Kronfeld et al. 1976]. It follows that the decrease in Ca absorption following parathyroidecmy observed in 123

6 124 McInsh and Tomas this experiment might be expected increase the rate of Ca removal from the bones. This compensary inflow of Ca the central pool would have maintained the plasma Ca levels, but for the increased net loss of Ca via the kidneys and intestinal secretion, as was observed. In conclusion, the data presented here have established that parathyroidecmy of sheep leads a reduction of P04 secretion and of Ca and P04 absorption. The effects on Ca were less consistent but in general an increased secretion of Ca seems have occurred. The nature of the response in Ca metabolism deserves further investigation. ACKNOWLEDGMENTS We gratefully acknowledge the skilled technical assistance of Ms. F. H. Bulman, Mr. M. T. Mano and Mr. D. D. Braybrook in undertaking this experiment and Mr. G. B. Belling for the radioactivity counting. REFERENCES BRONNER, F. (1964). Dynamics and Function of Calcium in Mineral Metabolism. Part 2A ed. Comar, C.L. and Bronner, F. Academic Press CALISLAR, T. and ST. CLAIR, L. E. (1974). Parathyroid glands of donmesticated ruminants. Journal of Dairy Science, 57, CARE, A. D., KEYNES, W. M. and DUNCAN, T. (1966). An investigation in the parathyroid origin of calcinin. Journal of Endocrinology, 34, CLARK, R. C., FRENCH, T. J., BEALE, A. M., CROSS, R. B. and BUTZ-OLSEN, 0. E. (1975). The acute effects of intravenous infusion of parathyriod hormone on urine, plasma and saliva in the sheep. Quarterly Journal of Experimental Physiology, 60, CLARK, I. and RIVERA-CORDERO, F. (1973). The effect of endogenous parathyroid hormone on calcium magnesium and phosphate metabolism in rats. Endocrinology, 92, KAY, R. N. B. (1966). The influence of saliva on digestion in ruminants. World Review of Nutrition and Dietetics 6, KEMM, J. R. (1976). The effect of parathyroidecmy and large doses of cholecalciferol on the ability of rats adapt changes in dietary intake of calcium. Journal ofphysiology, 256, KRONFELD, D. S., MAYER, C. P. and RAMBERG, C. F. (1976). Calcium homeostasis in cattle. In Handbook ofphysiology, vol. 7, Sec. 7. Ed. Greep, R. 0. and Astwood, E. B. (American Physiology Society, Washingn, D.C.) p MAYER, G. P., RAMBERG, C. F. and KRONFELD, D. A. (1967). Calcium metabolism and ]kinetics in intact and parathyroidecmized cows given parathyroid extract. Journal of Nutrition, 92, MAYER, G. P., RAMBERG, C. F. and KRONmELD, D. A. (1970). Parathyroid influences upon phosphorus balance and homeostasis in cows. Journal of Nutrition, 95, SAMMON, P. J., STACEY, R. E. and BRONNER, F. (1970). Role of parathyroid hormone in calcium homeostasis and metabolism. American Journal ofphysiology, 218, SHAH, B. G. and DRAPER, H. H. (1966). Depression of calcium absorption in parathyroidecmised rats. American Journal ofphysiology, 211, Tomas, F. M. (1974). The influence of parathyroid hormone on the secretion of phosphate by the parotid salivary gland of sheep. Quarterly Journal of Experimental Physiology, 59, TomAs, F. M., Momr, R. J. and SoMERs, M. (1967). Phosphorus turnover in sheep. Australian Journal of Agricultural Research, 18,