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1164 housed in clean cages, and placed in the animal care room. Ethical guidelines for animal care were followed. Effect of magnesium chloride and sodium fluoride on different Hyp fractions in rat kidneys The following groups of rats were studied (each consisting of 4-6 rats) (i) normal rats (Control group, n = 4 - 6 rats); (ii) rats injected with MgCl2 through intraperitoneal route 30 mg/kg body weight dose (MgCl2 treated group); (iii) rats injected with NaF through intraperitoneal route 10 mg/kg body weight dose (NaF treated group); (iv) rats injected with MgCl2 through intraperitoneal route 30 mg/kg body weight followed by NaF 10 mg/kg body weight through intraperitoneal route 30 minutes after MgCl2 injection (MgCl2 + NaF treated group). Preparation of the sample Rats were killed by carbon dioxide asphyxiation 24 hours after the final injection. The kidneys were dissected out, cleared of adhering tissues and weighed. The kidneys were then homogenized in normal saline (10% W/V) and the homogenate was used for Hyp determination as described below. Extraction of Free, Peptide- and Protein-bound Hydroxyproline Free and protein-bound Hyp was extracted by the method of Varghese et al. (1981) with slight modification. Briefly, 0.5ml of the homogenate was treated with 3 X 2 ml portion of re-rectified absolute alcohol and centrifuged at 3000rpm for 10min. The supernatants were pooled and evaporated to dryness. The residue was dissolved in suitable amount of distilled water and an aliquot of the extract was used for estimation of free Hyp. The peptidebound Hyp was determined after alkaline hydrolysis of the ethanol extractable fraction. The pellets were dissolved in distilled water and an aliquot of the extract was used for determination of protein-bound Hyp. The free Hyp fraction was measured on an aliquot of the ethanol extracted residue without alkali hydrolysis, whereas the peptide-bound Hyp was measured after alkaline hydrolysis. The precipitate obtained on ethanol treatment of the homogenate was subjected to alkali hydrolysis to determine protein-bound Hyp. Further details about the extraction of Hyp fractions have been described previously (Siddiqi et al., 2000). Hyp was determined in different fractions as described in the later section. Extraction of Soluble- and Insoluble-Collagen Hyp Soluble- and insoluble-collagen Hyp were extracted by the method of Kivirikko et al. (1965). Briefly, the tissue Canadian Journal of Pure and Applied Sciences samples were homogenized (4ml/g tissue) in a cold 0.45 M sodium chloride. The homogenate was extracted at 4 0 C for 24 hours with occasional stirring, followed by centrifugation at 13000rpm for 1hour. The supernatants obtained were precipitated with 4 volumes of a cold ethanol and were centrifuged twice with 80% ethanol, twice with absolute alcohol, twice with ether and twice with warm ethanol-ether (1:2). The residues were gelatinized with distilled water at 120 0 C for 3 hours and after filtration a sample of gelatine solution was used for soluble-collagen Hyp estimation as described below. The precipitates obtained after the above centrifugation were washed 3 times with 0.45 M NaCl and twice with distilled water, after which they were extracted with absolute ethanol, ether and ethanol-ether and gelatinized as above. A sample of gelatine solution was used for insoluble-collagen Hyp estimation as described in the following section. Determination of Hydroxyproline Concentration Hyp was measured by the modified alkaline hydrolysis method of Reddy and Enwemeka (1996). Briefly, an aliquot of the sample was added into NaOH (2 N final concentration) and the aliquot was hydrolyzed by heating in a boiling water bath for about 3-4 hours. An aliquot of 56 mM chloramine-T reagent was added to the hydrolyzed sample and oxidation was allowed to proceed at room temperature for 25min. Then an aliquot of 1M Ehrlich’s reagent (p-dimethylaminobenzaldehyde) was added to the oxidized sample and the chromophore was developed by incubating the samples at 65 0 C for 20 minutes. The absorbance was read at 550 nm using an Ultrospec 2000 UV/visible spectrophotometer (Pharmcia Biotech Ltd, Science Park, Cambridge, England). The Hyp concentration in the samples was calculated from the standard curve of Hyp. Further details about the optimization, linearity, specificity, precision and reproducibility of the method have been described previously (Siddiqi et al., 2000). Determination of Total Collagen Total collagen content was calculated from Hyp concentration assuming that Hyp constitutes 12.5% of total collagen (Edwards and O’Brien, 1980). Statistical Analysis Each sample was run in duplicate. The Hyp content was expressed as mean ± SD µg/gram wet tissue, for n = 4-6 rats. Hyp levels between groups were compared using one way ANOVA analysis followed by Tukey’s for multiple comparison test. Values were considered significant if p < 0.05. Statistical analysis was performed by means of InStat® package for personal computers (GraphPad TM Software, Inc., San Diego, USA).
RESULTS Table 1 shows the effect of MgCl2 and NaF on protein content of rat kidneys. MgCl2 and NaF alone caused a significant decrease of 70% (p
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