Characterization of intestinal phosphate absorption using a novel in vivo method

Katie Beth Williams, Hector F. DeLuca
2007 American Journal of Physiology. Endocrinology and Metabolism  
A new, completely in vivo method of measuring the rate of intestinal phosphate absorption has been developed. As expected from previous in vitro and ex vivo measurements, intestinal phosphate absorption is potently and rapidly stimulated by 1,25-dihydroxyvitamin D 3. The response is saturated with as little as 11.3 ng of 1,25-dihydroxyvitamin D 3 per day, consistent with a genomic mechanism. The effect of 1,25-dihydroxyvitamin D 3 disappears when the dosing solution of phosphate is at 2 M,
more » ... sting that 1,25-dihydroxyvitamin D 3 stimulates active transport of phosphate but not diffusion of phosphate. Finally, unlike findings resulting from in vitro or ex vivo experiments, no evidence in vivo was obtained that phosphate absorption requires sodium or is inhibited by potassium. vitamin D; phosphorus; intestine THE ACTIVE FORM OF VITAMIN D 3 , 1␣,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] is an integral regulator of calcium and phosphate homeostasis. In intestine, 1,25(OH) 2 D 3 is known to stimulate calcium and phosphate absorption (7). Whereas the effect of 1,25(OH) 2 D 3 on intestinal calcium absorption has been extensively studied, the effect of 1,25(OH) 2 D 3 on intestinal phosphate absorption is poorly understood. Previously, the role of 1,25(OH) 2 D 3 on intestinal phosphate absorption has been studied ex vivo, using everted intestinal segments or Ussing techniques, and in vitro, using brush border membrane vesicles isolated from intestinal tissue. Using these methods, several research groups have demonstrated that 1,25(OH) 2 D 3 increases intestinal phosphate absorption in chicks (14, 19) and rodents (1, 2, 4, 8, 10, 20, 21) . Early studies showed that everted intestine or intestinal segments in Ussing chambers require sodium for phosphate transport (3, 11) and that this is inhibited by excess potassium (3). More recent work suggests that this 1,25(OH) 2 D 3 -induced increase in intestinal phosphate absorption is mediated by a sodium-dependent phosphate cotransport protein type IIb, Na-P i IIb (4, 8, 21). However, the sodium dependence of phosphate absorption has not been demonstrated in vivo. Previous studies using everted intestinal segments or Ussing techniques followed intestinal phosphate absorption only in a small segment of the intestine ex vivo. However, the ability of 1,25(OH) 2 D 3 to increase phosphate absorption can vary greatly depending on the region of the intestine (10, 13, 20) . Furthermore, the rate of phosphate absorption in one segment of the intestine does not necessarily represent the overall rate of phosphate absorption along the entire length of the digestive tract in vivo. Even more troublesome is the use of brush border vesicles as representing transcellular transport. There appears to be a need for measurements of 1,25(OH) 2 D 3 -induced intestinal phosphate absorption in a live animal. In this study, we have developed a novel in vivo method for measuring intestinal phosphate absorption by using a radioisotope in rats based on a method previously described for measuring calcium absorption (12). With this method, we can clearly demonstrate a dosedependent increase in phosphate absorption in vivo in response to 1,25(OH) 2 D 3 . This response is rapid and occurs at low concentrations of phosphate where active transport is dominant and is absent at high concentrations where diffusion is dominant. This method does not confirm a dependence of phosphate absorption on sodium ions or an inhibition by potassium ions in clear contrast to previous results obtained with the ex vivo methods. MATERIALS AND METHODS Animals. Male weanling Sprague-Dawley rats (Harlan Sprague-Dawley, Madison, WI) were fed a purified vitamin D-deficient diet (18) for 8 wk ad libitum. Depletion of vitamin D stores was accelerated by alternating an adequate calcium diet with a calcium-deficient diet. Thus, during weeks 1, 5, and 8, the diet contained 0.47% calcium and 0.30% phosphorus. During weeks 2, 3, 4, 6, and 7, the diet contained 0.02% calcium and 0.30% phosphorus. All diets were supplemented with 100 l of soybean oil (Wesson oil; ConAgra Foods, Irvine, CA) containing 500 g of ␣-tocopherol, 60 g of menadione, and 40 g of ␤-carotene three times each week. Rats were housed in hanging wire cages under a UV-filtered 12:12-h light-dark cycle and had free access to distilled water. All experimental methods were approved by the Research Animal Resources Center at the University of Wisconsin-Madison. 1,25-dihydroxyvitamin D 3. 1,25(OH)2D3 was purchased from Tetrionics/Sigma-Aldrich (Madison, WI) and dissolved in 100% ethanol. The concentration of 1,25(OH) 2D3 was calculated according to Beer's law, using the UV absorbance at 264 nm and an extinction coefficient of 18,200 M Ϫ1 ⅐ cm Ϫ1 . 1,25(OH)2D3 was then dissolved in vehicle (5% ethanol, 95% propylene glycol). Vehicle (0.1 ml) containing as much as 180 ng of 1,25(OH) 2D3 was injected intraperitoneally each day for up to 5 days. Serum analysis. When rats were injected daily for 5 days, blood was collected from the tail artery under ether anesthesia before the first and fifth injections for serum analysis. When rats were injected only once, blood was collected before the injection in the same manner. Blood was centrifuged at 1,500 g for 15 min at 22°C to yield serum. The serum calcium level was determined by flame atomic
doi:10.1152/ajpendo.00654.2006 pmid:17299082 fatcat:ck6wx4ibajgirkwtlxbwamqufq