EFFECTS OF LOW CALCIUM AND LOW PHOSPHORUS DIETS ON THE DUODENAL ABSORPTION OF CALCIUM IN BETAMETHASONE-TREATED CHICKS

J. FOX, A. D. CARE AND J. BLAHOS Department of Animal Physiology and Nutrition,

University of Leeds, Leeds, LS2 9JT

(Received 9

December

1977)

SUMMARY

The effect of oral administration of betamethasone (25 \g=m\gkg\m=-\1day\m=-\1) on the duodenal absorption of calcium has been studied in chicks using the ligated loop technique in vivo. The chicks were fed normal calcium, normal phosphorus (NCaNP), low calcium, normal phosphorus (LCaNP) or normal calcium, low phosphorus (NCaLP) diets. Daily oral administration of betamethasone for 2\p=n-\3weeks markedly reduced the absorption of calcium in chicks fed the NCaNP diet, but did not significantly affect the adaptation in absorption when the NCaLP or LCaNP diets were fed for the same period of time. In one group of chicks, betamethasone was administered daily for 10 days before the birds were transferred to the NCaLP or LCaNP diets. Adaptation was again unaffected by betamethasone treatment. Administration of betamethasone caused a marked retardation in growth-rate, hypercalcaemia and an increased percentage of ash in the tibiae. INTRODUCTION

long-term administration of glucocorticoids has been shown to reduce the intestinal absorption of calcium in vivo (Collins, Garrett & Johnston, 1962; Fox, Care & Marshall, 1978) and to inhibit the active transport of calcium from the small intestine in vitro (Harrison & Harrison, 1960). Cholecalciferol (vitamin D3) is activated by two successive hydroxylations ; the first, at position 25, occurs primarily in the liver; the second, at position 1, is achieved in the kidney. 1,25-Dihydroxycholecalciferol (l,25-(OH)2CC) stimulates the intestinal transport of calcium and induces the synthesis of a calcium-binding protein (CaBP) in the intestinal mucosa. The increase in the efficacy of absorption of calcium in response to the feeding of diets low in calcium or phosphorus (Morrissey & Wasserman, 1971) is brought about by an increase in the renal production of l,25-(OH)2CC (Omdahl, Gray, Boyle, Knutson & DeLuca, 1972; Baxter & DeLuca, 1976). Low concentrations of phosphate in the plasma also enhance the uptake of l,25-(OH)2CC by the intestinal mucosa (Friedlander, Henry & Norman, 1977). Initially it was postulated (Harrison & Harrison, 1960) that glucocorticoids inhibit the intestinal absorption of calcium by antagonizing the action of vitamin D. However, the renal production of l,25-(OH)2CC, its tissue distribution and subcellular localization on or in the intestinal mucosal cell nuclei have been shown to be unaffected in rats treated with cortisone (200 mg kg-1 day-1) for 7 days (Kimberg, Baerg, Gershon & Graudusius, 1971 ; Fa vus, Kimberg, Millar & Gershon, 1973). Furthermore, Lukert, Stanbury & Mawer (1973) showed an inhibition of absorption of calcium from the small intestine in vitro, despite an The

Present address: Czechoslovakia.

Faculty of Paediatrics, Department of Medicine, Charles University, Prague,

increased concentration of l,25-(OH)2CC in the serum of prednisolone-treated rats. The concentration of CaBP in the intestinal mucosa of glucocorticoid-treated rats has been shown to be normal (Krawitt, 1972) or higher than the control value (Kimberg et al. 1971). This study was designed to explore the possibility that the intestinal absorption of calcium could be inhibited by the oral administration of betamethasone, a synthetic glucocorticoid, at a dose rate commonly found in veterinary medicine (25 µg kg-1 day-1) and also to measure any change in the increase in efficacy of absorption of calcium caused by dietary restriction of calcium or phosphorus which might be associated with betamethasone administration. MATERIALS AND METHODS

One-day-old chicks (Thornbers Type 909; Mytholmroyd Hatching Co., Hebden Bridge) kept in a thermostatically controlled room under conditions of continual light. They offered a commercial chick starter diet (Linton Mill (Wintringham) Ltd, Maltón) containing 1% calcium and 1-1% phosphorus together with water ad libitum. were were

Experiment 1 After 8 days the chicks were divided into three weight-matched groups. One group was fed a normal calcium, normal phosphorus (NCaNP) semi-synthetic diet (1-2% Ca, 0-65% P, 1000 i.u. vitamin D3/kg), another group was fed a low calcium, normal phosphorus (LCaNP) diet (0-1% Ca, 0-65% P, 1000 i.u. vitamin D3/kg) and the third group was fed a normal calcium, low phosphorus (NCaLP) diet (1-2% Ca, 0-25% P, 1000 i.u. vitamin D3/kg). The composition of the diets was similar except for the calcium or phosphorus content and was similar to those described by Morrissey & Wasserman (1971). Half the chicks from each dietary group received a daily oral dose of betamethasone (Betsolan Injection; Glaxo, Greenford) at a rate of 25 µg kg-1 day-1. The betamethasone was administered in 0-5 ml water by a stomach tube. Control birds were untreated. After 14 days of treatment the duo¬ denal absorption of calcium was measured as described below.

Experiment 2 The conditions were similar to those described in experiment 1. The chicks were divided into three weight-matched groups at 16 days of age and transferred to the NCaNP, LCaNP and NCaLP diets. Half the chicks from each dietary group received a daily oral dose of beta¬ methasone (25 µg kg-1 day-1) in water. Control birds were untreated. After 21 days of treat¬ ment the

absorption of calcium from the duodenum was measured. Experiment 3

The chicks were divided into three weight-matched groups at 6 days of age and half of the birds from each group were orally dosed with betamethasone (25 µg kg-1 day-1) as above for 10 days, during which time the commercial diet was fed. Control birds were untreated. The chicks were then transferred to the NCaNP, LCaNP and NCaLP diets and betametha¬ sone treatment was continued. After a further 11 days the absorption of calcium from the duodenum was measured. Measurement

of calcium absorption

The chicks were fasted overnight but allowed access to water and after the weight of each chick was recorded, the absorption of calcium from the duodenum was determined in vivo by the ligated loop technique (Morrissey & Wasserman, 1971). Each chick was anaesthetized with diethyl ether, the duodenum was exteriorized and a ligature tied just below the gizzard. Another ligature was placed loosely around the other end of the segment approximately 5 cm long. A hypodermic needle was inserted through the tie into the intestinal lumen and after the ligature was tightened around the needle, a solution containing 150 mM-NaCl,

25 mM-Ca2+ and 0-2 µ 47Ca/ml, pH 7-0, was injected through the needle into the lumen. The time of injection was noted and the segment was replaced in the abdominal cavity. The incision was closed with wound clips. As the betamethasone-treated chicks were smaller than the controls, care was taken to ensure that the length of the segment was similar in the chicks from each group. After 30 min a blood sample was taken by cardiac puncture and the bird was killed by an intracardiac injection of sodium pentobarbitone. The ligated segment was excised and placed in a tube for measurement of the residual 47Ca activity. The right tibia was removed and cleaned of all muscle tissue. The bone was placed in a paper thimble and subjected to lipid extraction with ether for two 8 h periods. The fat-free bones were dried overnight at 100 °C, cooled in a desiccator and weighed. The bones were then converted to ash overnight at 480 °C in a muffle furnace, cooled in a desiccator and reweighed. The percentage of ash in each fat-free bone was calculated. The concentrations of calcium and inorganic phosphate in the plasma samples were determined by automated methods (Technicon methods N-3b and N-4b respectively). Radioactivity (47Ca) remaining in the duodenal loop was measured using an automatic welltype scintillation counter with a sodium iodide crystal (Gammaguard 150; ICN Tracer Laboratories, Weybridge). A pulse-height analyser was used to exclude emissions from 47Sc, the daughter isotope of 47Ca. RESULTS

The results from the three experiments are shown in Table 1. In each experiment, the oral administration of betamethasone caused a highly significant inhibition of duodenal 47Ca absorption in chicks fed the NCaNP diet. Feeding the LCaNP or NCaLP diets resulted in the expected stimulation of absorption of calcium in the control chicks. Moreover, the administration of betamethasone had no significant effect on this adaptation, even when the results from all three experiments were combined. There was always a significant increase in the concentration of calcium in the plasma and usually a tendency for the concentration of phosphate to fall in the betamethasone-treated chicks compared with controls, irrespective of which diet was fed. Hypocalcaemia and hypophosphataemia were observed in control chicks fed the LCaNP and NCaLP diets. The administration of betamethasone caused a significant increase in the percentage ash in the tibia under all dietary conditions except for the NCaNP diet in experiment 1. The increase was more marked in the tibiae of the chicks fed the LCaNP or NCaLP diets, in which animals the percentage of ash was lower than in those fed the NCaNP diet. In all experiments the administration of betamethasone significantly retarded the growth of the chicks whichever diet was fed. The rate of growth was usually lower in the control chicks fed the LCaNP and NCaLP diets compared with the control chicks fed the NCaNP diet. DISCUSSION

The

feeding

of diets low in calcium or in phosphorus has been shown to increase the of calcium from a ligated loop of duodenum (Morrissey & Wasserman, 1971). This was confirmed in the present study. The oral administration of betamethasone, at a rate commonly used in veterinary medicine (25 µg kg-1 day-1), for 2 or 3 weeks, significantly reduced the absorption of calcium in chicks fed the NCaNP diet but had no effect on the increased percentage absorption seen when either the low calcium or low phosphorus diet was fed. These results are difficult to reconcile with those of Winter, Morava & Simon (1969) who showed that the administration of cortisone (0-5 mg/day) for 2 weeks to young rats had no effect on the duodenal or jejunal absorption of calcium when they were fed a normal (0-6%) calcium diet, but prevented the increase in absorption of calcium when a low (0-1%) calcium diet was provided.

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