M.L. Brance

Luminal calcium concentration controls intestinal calcium absorption by modification of intestinal alkaline phosphatase activity.

Intestinal alkaline phosphatase (IAP) is a brush-border phosphomonoesterase. Its location suggests an involvement in the uptake of nutrients, but its role has not yet been defined. IAP expression parallels that of other proteins involved in Ca absorption under vitamin D stimulation. Experiments carried out in vitro with purified IAP have demonstrated an interaction between Ca and IAP. The gut is prepared to face different levels of Ca intake over time, but high Ca intake in a situation of a low-Ca diet over time would cause excessive entry of Ca into the enterocytes. The presence of a mechanism to block Ca entry and to avoid possible adverse effects is thus predictable. Thus, in the present study, Sprague-Dawley rats were fed with different amounts of Ca in the diet (0.2, 1 and 2 g%), and the percentage of Ca absorption (%Ca) in the presence and absence of L-phenylalanine (Phe) was calculated. The presence of Phe caused a significant increase in %Ca (52.3 (SEM 6.5) % in the presence of Phe v. 31.1 (sem 8.9) % in the absence of Phe, regardless of the amount of Ca intake; paired t test, P = 0.02). When data were analysed with respect to Ca intake, a significant difference was found only in the group with low Ca intake (paired t test, P = 0.03). Additionally, IAP activity increased significantly (ANOVA, P < 0.05) as Ca concentrations increased in the duodenal lumen. The present study provides in vivo evidence that luminal Ca concentration increases the activity of IAP and simultaneously decreases %Ca, acting as a minute-to-minute regulatory mechanism of Ca entry.

Effect of calcium on rat intestinal alkaline phosphatase activity and molecular aggregation

Two fractions of rat intestinal alkaline phosphatase (IAP) were detected by Western blot: 168 ± 6 and 475 ± 45 kDa. The low molecular weight fraction constitutes 43% of the isolated proteins exhibiting 82% of the enzymatic activity, and a heavier fraction constitutes 57% of the isolated proteins and has 18% of the enzymatic activity. Calcium produced an increase of the 475-kDa form to the detriment of the 168-kDa form. This work also describes the kinetic and structural changes of IAP as a function of calcium concentration. With [Ca2+] < 10 mmole/L, the Ca2+-IAP interaction fitted a binding model with 7.8 ± 4.4 moles of Ca2+ /mole of protein, affinity constant = 19.1 ± 8.4 L/mmole, and enzymatic activity increased as a linear function of [Ca2+] (r = 0.946 p < 0.01). On the other hand, with [Ca2+] >10 mmole/L the data did not fit this model and, the enzymatic activity decreased as a function of [Ca2+] (r = − 0.703 p < 0.05).

Methodology developed for the simultaneous measurement of bone formation and bone resorption in rats based on the pharmacokinetics of fluoride

This paper describes a novel methodology for the simultaneous estimation of bone formation (BF) and resorption (BR) in rats using fluoride as a nonradioactive bone-seeker ion. The pharmacokinetics of flouride have been extensively studied in rats; its constants have all been characterized. This knowledge was the cornerstone for the underlying mathematical model that we used to measure bone fluoride uptake and elimination rate after a dose of fluoride. Bone resorption and formation were estimated by bone fluoride uptake and elimination rate, respectively. ROC analysis showed that sensitivity, specificity and area under the ROC curve were not different from deoxypiridinoline and bone alkaline phosphatase, well-known bone markers. Sprague–Dawley rats with modified bone remodelling (ovariectomy, hyper, and hypocalcic diet, antiresorptive treatment) were used to validate the values obtained with this methodology. The results of BF and BR obtained with this technique were as expected for each biological model. Although the method should be performed under general anesthesia, it has several advantages: simultaneous measurement of BR and BF, low cost, and the use of compounds with no expiration date.