Determination of calcium in feces and urine samples

The procedure described by Kansal and Chaudhary (1982) was used. The pooled fecal material from each rat collected during the 7-day balance period was dried overnight at 102 ± 2 °C. The dried feces (W) were weighed and ground into a powder with a pestle and mortar and kept in air-tight plastic containers in a refrigerator until required for analysis. An accurately weighed quantity, w (0.6 g) of each sample was charred in a silica dish, cooled and ashed overnight in a muffle furnace at 525 ± 5 °C, followed by cooling in a dessicator, weighing and adding to it 10 ml of 6 N HCl (Ranganna 2001). This solution was heated to dryness using an electrical heating coil (Bajaj Electricals). Fifteen milliliters of 3 N HCl were carefully added to it avoiding splashing. The solution was heated just enough to bring to boil and then cooled and filtered through Whatman No. 41 ashless filter-paper into a 100 ml volumetric flask (prerinsed with distilled water and 3 N HCl). Further 10 ml of 3 N HCl was added to the same silica dish and heated until the solution just began to boil, followed by cooling and filtering through the same filter-paper into the volumetric flask. The dish was washed thrice with distilled water, and the washings were collected into the same volumetric flask after passing through the filter paper. To the washings collected along with the filtrate, 0.5 ml of 7.5% of the EDTA (disodium salt) solution was added. The flask content was cooled and its volume made up to 100 ml with distilled water. The absorbance of samples was measured with an atomic absorption spectrophotometer (at a wavelength of 422.7 nm) and the concentration of calcium (ppm) was estimated from the standard curve. A blank determination was carried out following the same procedure without sample. The calcium concentration (in ppm) of the sample was converted to mg calcium present in the total fecal material using the following formula:

where a, concentration of calcium in the sample solution, µg/ml; b, concentration of the calcium in the sample blank, µg/ml; v, final volume made up of the ash solution or digest of the sample, ml; w, weight of the fecal sample taken for analysis, g.

Therefore,

The urine from individual rats was collected daily into separate glass bottles by carefully rinsing with toluene, the funnel and the attached urine-collecting plastic container of the metabolic cage. The collection was made for the balance period of 7 days. The pooled urine from each rat was carefully transferred to a silica dish in installments and evaporated on an electrical heating coil (Bajaj Electricals). To ensure quantitative transfer of the material each glass bottle was rinsed twice with toluene (10 ml) and deionised distilled water (10 ml). It was then evaporated to dryness. The dried material was then ashed overnight in a muffle furnace at 525 ± 5 °C. The ash quantity was determined and its calcium content estimated in the same way as for ash of the fecal samples above. A blank was also run simultaneously and calcium quantity worked out as

where a, concentration of the calcium in the sample solution, µg/ml; b, concentration of the calcium in the sample blank, µg/ml.

The absorption and retention of calcium by experimental animals were calculated as follows:

Calcium intake was calculated from the measured amount of calcium present in a diet and the amount of that diet consumed by a rat.

The data obtained was statistically analyzed for variance through the analysis tool in ANOVA using Excel-2000 (Microsoft).