BONE COMPOSITION AND BIO-AVAILABILITY OF CALCIUM IN YOUNG WEANLING RATS FED DIETS OF VARYING SOURCES OF CALCIUM AND PROTEIN

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1 J. Dairying, Foods & H.S. 24 (3/4) : , 2005 BONE COMPOSITION AND BIO-AVAILABILITY OF CALCIUM IN YOUNG WEANLING RATS FED DIETS OF VARYING SOURCES OF CALCIUM AND PROTEIN J.J. Dhaduk, 5.5. Sail and S. Rema Post-Graduate Department of Home Science, Sardar Patel University, Vallabh Vidyanagar , Gujarat. India ABSTRACT Bone composition and bio-availability of calcium (289mg %) from egg shell, Oyster shell and vegetable mixture with variable quality of proteins (log %) was studied after 4 weeks of experimental feeding in 25 young albino rats weighing g. Fresh femoral and tibial weight, length, moisture, dry weight and fat-free dry weight were observed highest in egg protein Gr IV and lowest in vegetable protein Gr V as compared to control Gr I. The egg shell was also found to be better than Oyster shell in this respect. In contrast, both femoral and tibial fat levels were high in Gr V (12.61 and 15.23g %) and very low in Gr IV (3.50 and 4.50g %) as compared to Gr I (8.29 and 9.45g %) respectively. Femoral and tibial (fresh weight basis) calcium and phosphorus levels were highest in Gr IV (8.82 and 2.29g %, 9.71 & 2.89g % respectively), higher in Gr IV and high in casein plus Oyster shell Gr II when compared with Gr lor casein plus egg shell Gr III. Femoral iron content was highest in Gr V (9.39mg %) and tibial iron in Gr II (8.46mg %). Further the calcium and phosphorus levels were lower and iron content was higher in femur than in tibia. Femoral (fat-free dry bone),ash weight was highest in Gr IV (50.23g %) and lower in other groups as compared to Gr I (49.85g %), while femoral ash weight in Gr II (49.34g %) was higher than in Gr III (48.18g %). Femoral calcium, phosphorus and iron levels were greater in Gr V compared to all other groups. Iron level was moderately good in casein plus Oyster shell Gr II. The tibial (fat-free dry bone) ash and calcium levels were greater in Gr V (52.74 and 17.91g %) respectively, phosphorus in Gr IV (5.26g %) & iron in Gr II (16.98mg %). Casein plus Oyster shell showed better results than casein plus egg shell group in respect of tibial ash and mineral contents. It is concluded that the calcium bioavailability was the best in egg protein as compared to casein and vegetable proteins. There was no difference in available calcium from Oyster shell and egg shell as compared to salt mixture used in the experiment... INTRODUCTION The skeleton provides a solid frame to the body and gives support tothe soft tissues. The skeleton constitutes 16%of.the whole body and more than 99% calcium is deposited as calcium -phosphate within the skeleton and / teeth, it is not surprising that bone tissue act as a calcium reservoir, and, at least in rats, is sensitive to dietary factors (Boskey, 1988). Though somewhat variable according to age, state of nutrition, quality of diet and species, the normal adult bone is composed of approximately 45% water, 25% ash, 20% protein and 10% fat. In mammals, the ash is accounts for 36% caftium, 17% phosphorus (Teegarden et a/., 1998). The amount of calcium present in bone depends on level of calcium in the diet (Thomas et a/., 1988; Peterson et a/., 1992). The importance of lipids in the maturation of the bone was considers with 4-6 fold increase in lipid of the epiphyses. The presence of phospholipid acts as transport of phosphorus and calcium. Dietary protein is one of the important factors involved in normal bone development. Kerstetter et a/. (2003) reported that quality of the dietary protein affect growth and mineralization of long bone. Bone is complex tissue that changes slowly. As such, it is difficult to conduct well-controlled nutrition studies to quantify the effect of one nutrient on bone (Promislow, 2002). The bones serve not only as structural elements but also as storehouse of calcium and phosphorus, which may be mobilized at timeswhenthe assimilation ".~.

2 of these minerals is inadequate to meet the body needs, The water content of bone decreases with age while fat is varies according to the nutritive state, An attempt was, therefore, made to study the bone composition and bio-availability of calcium (289 mg %) from egg shelj, Oyster shell and from vegetable mixture with changing quality of proteins (log %) over a period of 4 weeks in young albino rats, MATERIAL AND METHODS This study was carried out over a period of 4 weeks on 25 young albino weanling rats weighing 60,9-63.3g divided at random into 5 groups, each of 5 animals. The rats were caged individualjy in galvanized iron cages and were fed and watered ad-libitum, The composition of diet fed to different groups of experimental animals was as given in Table 1, The salt/vitamin mixtures and vitaminised oil incorporated in the ration were prepared according to Oser (1976). The raw materials used in preparation of ration were procured from the local market of Anand city. The content of food nitrogen was estimated by macro-kjeldahl method (Oser, 1976) and was multiplied by a factor 6.25 (for casein 6,38) to obtain g% value of crude protein in different raw ingredients and diets. The calcium, phosphorus and iron contents of raw ingredients, diets and bones were estimated according to titrimetric method of Clark and Collip (1925) and colorimetric methods of Fiske and Subba Row (1925) and Ramsay (1953), respectively, using ash content of materials. At the end of 4 weeks of feeding experiment, rats were etherized in desiccators using pure diethyl ether. Bones (femur and tibia) were removed, cleaned, weighed and then oven dried at 60 C to determine moisture.content and subsequently the total lipids and bone ash content according to AOAC (1984). Vol. 24, No. 3/4, The length of oven-dried bones was measured by the vernier caliper. The data were analyzed statisticaljy (Gupta, 1984). RESULTS AND DISCUSSION The mean fresh weight, moisture content and length of femoral and tibial bones of rats fed different experimental diets were as given in Table 2. Gr I, II and III diets contained same quality and quantity of protein (log % casein), but the source of calcium was different. The fresh weight of left femur and tibia ranged from to 0.504g and to 0.423g, respectively, were not significant differences among the 3 groups. Both femoral and tibial fresh weight of Gr I and III animals were lower as compared to weights of control Gr I. This indicated that some of the elements/substances present in Oyster shelj and egg shelj powder which suppress the bone weight. Diets contained same amount of calcium and protein in Gr I, IV and V, but the source of protein was different i.e. casein, egg protein and vegetable protein, respectively. Both the femoral and tibial weights of Gr IV animab were the highest and those of Gr V were the lowest. The differences may be attributed to quality of protein present in the diet. Gr IV diet contained good quality egg protein, may have affected the bone growth. Kerstetter et al. (2003) reported that growth and mineralization of long bone affected by quality of dietary protein. Gr V diet contained poor quality vegetable protein and some anti-nutritional factors - phytate and oxalate, which affecting adversely the bone growth (Standsteat, 1992). Gr I diet contained moderately good protein; therefore bone weight was more than that in Gr V. The femoral and tibial development was proportionate to the quality of protein as the quality of protein increases the fresh weight also increases. Femoral and tibial moisture content of Gr I, II and III animals was in very narrow range and did not show significant differences, but the tibial moisture content was significantly

3 206 J. DAIRYING, FOODS & H.S. Table 1. Composit!on of diets fed to different groups of experimental :'ats S.No. Ingredients C;r I GrII GrIll GrlV GrV 1. Starch (Sago) (g) Casein (g) Egg powder (g) Ragi (g) Wheat (g) Bengal gram (g) Salt mixture (g)* Salt mix without calcium (g) Vitamin mixture (g)' Vitaminized oil (g)' Oyster shell powder (g) Egg shell powder (g) Total As per Oser (1976). $ See Table 2 $ As per Oser (1976) but omitted calcium citrate 308.2g, calcium biphosphate 112.8g and calcium carbonate 68.5g from the mixture which was replaced by starch 489.5g. Gr! Basal diet (log % casein proteins). Gr II Basal diet with salt mix without calcium m~ calcium from Oyster shell powder. Gr III Basal diet with salt mix without calcium mg calcium from egg shell powder. Gr N Casein of basal diet replaced by egg powder (log % proteins) and salt mix without calcium mg calcium from egg shell powder. Gr V Vegetable protein (log % proteins: ragi-wheat-bengai gram mix) and salt mix without calcium mg calcium from vegetable and egg shell powder. Table 2. Fresh weight, length and moisture content of left femur and Group no. Left Femur Left Tibia Fresh wt. (g) Length (em) Moisture (g %) Fresh wt. (g) Length (em) Moisture (g %) I O.50±O ±0:O ± ± ± ±1.53 II 0.46±O ±0.O ±1.53 O.40± ± ,75±1.10 III O,47±0,OZ Z,83± ± ±O ± ±0.05 IV 0.54±O.02* 3.0Z±O ± ± ±0.04** 4Z.83±0.63 V O.4Z±O.OZ* Z,72± ±0.4Z 0.34±O ± ±0.57 * Significant at 5 per cent level. Significant at 1 per cent level. lower in Gr III compared to Gr I and II. Further, the femoral and tibial moisture content of Gr I, IV and V ranged between to and to 42.83g%, respectively. Gr V showed significantly (P<0.05) lower values of tibial moisture content compared to control Gr I were attributed to the quality of protein (Zemel, 1988). Gr I, IV and V diets contained moderately good, good and poor quality proteins, respectively. The moisture content of femur was higher than that of tibia, which contained less of cortex, and thereby less moisture. Sharma (1983) and Pandya (1990) also obtained similar results. No significant differences were observed among the group of I, II and III for femoral and tibial length of rats. Among the Gr I, IV and V femoral length was significantly highest (P<O.Ol) in Gr IV (3.02cm ± 0.06)

4 Vol. 24, No. 3/4, and lowest (P<0.05) in Gr V (2. 72cm ±.0.03). The same trend shown in tibial length, the overall tibial length was greater than femoral length, bone length depend on type of bone. Further it has shown the same trend like fresh weight of femur and tibia in different groups. Bone length is dependent on growth of animal and quality of protein indirectly is responsible for growth (Hannan et ai., 2000). Gr IV diet contained good quality protein, therefore it showed high values for bone length than Gr I and V. Yuan and Kitts (1991) confirmation that in poor quality protein femoral and tibial bone growth was suppressed in rats. The mean values for the femoral and tibial dry weight, fat-free dry weight and fat content (dry weight basis) are shown in Table 3. Femoral and tibial dry weight of animals in Gr I, II and III diets ranged from to and to g, respectively. The values in Gr II and III were relatively lowerthan control Gr I. Results suggested that salt mixture added in the diet Gr I showed better bone formation as compared to Oyster shell and egg shell powder. In contest of both femoral and tibial weight of egg protein Gr IV showed highest and vegetable protein Gr V iqwest. The femoral and tibial dry weights of Gr I, IV and V ranged from to and to g, respectively, Gr V being significantly (P<0.05) lower in femoral and tibial dry weight than Gr IV and I. It further proved that the development of bone is enhanced by the quality of protein (Cooper et al., 1996). Femoral and tibial fat-free dry weights of Gr I, II and III animals varied from to and to g, respectively, and theirfat/lipid levels ranged from 8.03 to 8.37 g% and 9.45 to g%, respectively on dry weight basis. No significant differences were observed in femoral fat or fat-free dry weight among the three groups. The fat-free dry weight of both the bones was significantly (P<O.OI) higher and lower in egg protein Gr IV and vegetable protein Gr V, respectively as compared to control, but the trend of fat content was inverse being significantly lowest in Gr IV (P<0.01) and highest in Gr V (P<0.05l. It may be due to quality of protein as some of substances present in egg protein that may reduce the lipid levels of bone. While Gr V diet contained vegetable origin protein, which may have some substances favouring the high lipid level of bone. Femoral and tibial calcium. phosphorus and iron levels on fresh weight basis are presented in Table 4. No significant differences were observed in femoral and tibial calcium and phosphorus among the casein protein Gr I, II and III, but tibia phosphorus in egg and vegetable protein Gr IV and V were significantly (P<0.05) higher than casein protein Gr I. Unlike femur, calcium and phosphorus contents of tibia were higher and the significant variation found between groups in tibial mineral contents was difficult to explain. Bone composition differs according to age, diet and even from bone to bone. The femoral iron content in none of the groupswas significantly different, but tibial iron content in Gr IV was significantly (P<0.05) lower than in Gr II. The femoral and tibial iron content in Gr I, IV and V were none significantly difference, but femoral high bone iron seen in vegetable protein Gr V. Tibial high iron seen in Oyster shell Gr II it may be due to some unknown compound present in Oyster shell which enhance the iron absorption. It was interesting to note that femur contained more of iron as compared to tibia and it was exactly reverse in case of calcium and phosphorus. The absolute calcium, phosphorus and iron levels per femur and per tibia are shown in Table 5. No significant differences were observed in femoral calcium in Gr I, II and III. While tibial calcium Gr III was lower than in Gr I. As calcium (mg/bone) values depend on bone growth, Gr II and III animals showed low growth I

5 208 Group no. J. DAIRYING, FOODS & H.S. Table 3. Dry weight, fat-free dry weight and fat Contents of left femur and Left Femur Left Tibia I II III IV V Drywt. (g) Fat free dry wt. (g) Fat content (9 l){l) Drywt. (g) Fat free dry wt. (g) Fat content (g %) 0.27± ±0.Ol 8.29±1.56 O.24±O.Ol O.22±OOl 9.45±1.60 O.25±O.Ol O.23±O.Ol 803±O.71 O.23±O.Ol O.20±O.Ol 11.32±1.21 O.25±O.Ol O.23±O.Ol 8.37±O.82 O.22±O.Ol O.20±OOl 10.39±2.31 O.29±O.Ol O.28±O.O 1* 3.50±O.24** O.24±O.Ol O.23±O.Ol* 4.50±O.93*' O.23±O.Ol* O.20±O.Ol" 12.61±2.00" O.21±O.Ol' O.18±OOO* 15.23±O.96', Significant at 5 per cent level. " Significant at 1 per cent level. Table 4. Mineral contents of left femur and tibia of rats fed different experimental diets (fresh weight) (mean ± SE) Group no. Left Femur Left Tibia Calcium (g %) Phosphorus (g %) Iron (mg %) Calcium (g %) Phosphorus (g %) Iron (mg %) I 8.35±O.1l 212±O ±O ±O ±O.Ol 6.80±O.66 II 8.27±O ±O ±O ±O ±O ±O.43 III 8.32±O ±O ±O ±O ±O ±O.25 IV 8.82±O ±O ±O ±O ±O.10' 6.98±O.24' V 8.51±O ±O ± ±O ±O ±O.14, Significant at 5 per cent level. Table 5. Absolute mineral contents (mglbone) of left femur and Group no. Left Femur Left Tibia Calcium Phosphorus Iron Calcium Phosphorus Iron I 41.81±O ±O.23 O.O4±O.Ol 38.30±O ±O.32 O.O3±O.OO II 38.07± ±O.29 O.O4±O.OO 35.06± ±O.60 O.O3±O.OO III ± ±O.27 O.O4±O.OO 34.93± ±O.47 O.O3±O.OO IV 47.59±2.46' 12.13±O.76' O.O5±O.OO 41.37± 1.33' 12.33±O.50' O.O3±O.OO V 35.25±O.56" 9.23±O.43 O.O4±O.Ol 33.05±O.71" 9.60±O.33 O.O3±O.OO, Significant at 5 per cent level. " Significant at 1 per cent level. rate compared to Gr I, presumably because of lower bone calcium. The values of femoral and tibial calcium were significantly higher ingr IV and lowest in Gr V (P<0.01). Deposition of calcium in bone depends on the quantity of dietary protein (Patel, 1986; Poneros-Schne:r et al., 1989 and Kerstetter et al., 2003). Significant (P<0.05) differences were observed in phosphorus values among casein protein group Gr II and III compared to control Gr I. Phosphorus content of femur and tibia in Gr IV was significantly higher than in Gr I and V (P<0.05). In the present study, phosphorus levels were proportionate to the quality of protein fed to animals. Femoral and tibial values of iron per bone in all groups were practically same Le mg/bone and 0.03 mg/bone, respectively. Femoral and tibial ash, calcium and phosphorus levels on dry and fat-free dry

6 Vol. 24, No. 3/4, Table 6. Ash and mineral contents of fat-free dry left femur and Fat free dry Left Femur Fat free dry Left Tibia Calcium (g 'Yo) Phosphorus (g 'Yo) Iron (mg %) 1682± ± ± ± ± ± ± ± ± ± ± ±1.35 Ash (g 'Yo) 5158± ± ± ±0.34 Calcium (g %) Phosphorus (g %) Iron (mg%) 17.56± n±0.1l 13.20±l ± ± ±130' 17.38± ± ± ± ± ±036 V 4909± ± ± ± ±0.61 n = 5 rats in each group. Read significance column wise. Significant at 5 per cent level. 1791± ± ±0.43 weight basis were nearly identical among the five groups (Table 6). Animals fed egg protein Gr IV showed little higher bone ash as compared to those fed vegetable protein. Gr V showed highest values for calcium, phosphorus and iron but none of significantly different. As seen in earlier table, fat and moisture were high in this group. When they were removed, the ratio changed a little and showed higher values than control and other experimental groups. Femoral and tibial iron levels of Gr II and V showed high values as compared to other groups. It is possible that unknown compounds present in Oyster shell (Gr II) might have enhanced the iron intake, absorption and deposition in bone. High bone value is probably due to cumulative effect of both the factors mentioned above. From these results it is concluded that the calcium bioavailability is best in egg protein compared to casein and vegetable protein. Vegetable protein retards the bone growth in animal but calcification and mineralization was same as casein and egg protein. As far as calcium source is concern, there was no difference in available calcium from Oystershell and egg shell powder compare to salt mixture used in this experiment. REFERENCES AOAC (1984). Official Methods ofanalysis. 14th edn., Association of Official Analytical Chemists, Washington, D.C. Boskey, A.L. (l988). In: Calcium and Human Biology (Nordia, B.E.C. ed.). Springer- Verlag, Berlin and Heidelberg, Germany, pp Clark, E.P. and Collip, J.B. (1925). J. Bioi. Chem., 63: 461. Cooper, C. et al. (l996). Calcif Tissue Int., 58: Fiske, C.H. and Subba Row, Y. (1925). J. Bioi. Chern., 66: Gupta, S.C. (1984). Fundamentals of Statistics. 1st edn., Himalaya Publishing House, Bombay. Hannan, M.T. et al. (2000). J. Bone. Miner Res., 15: Kerstetter, J.E. et al. (2003). Am. J. Clin. Nutr., 78: 584s-592s. Oser, B.L. (1976). Hawk's Physiological Chemistry. 14th edn., T.M.H. Publishing Company Ltd., New Delhi, pp Pandya, B.N. (1990). M.Sc. Thesis, S.P. University, Vallabh Vidyanagar. Patel, V.H. (1986). M.Sc. Thesis, S.P. University, Vallabh Vidyanagar. Peterson, C.A. et al. (1992). J. Nut., 122: Poneros-Schneier, A.G. and Erdman, J.w. Jr. (1989). J. Food Sci., 54{l): Promislow, J.H. et al. (2002). Am. J. Epidemiol., 155: Ramsay, W.N.M. (1953). J. Bioi. Chern., 53: 227. Sharma, A. (1983). M.Sc. Thesis, M.S. University, Baroda. Standsteat, H.H. (l992). Nutr. Rev., 50:

7 210 J. DAIRYING, FOOD5 & H. S Teegarden, D. et al. (1998). Am J. Clin. Nutr., 68: Thomas, M.L. et al. (1988). J. Bone and Minerals, 4: Yuan, Y.Y. and Kitts, D.o. (1991). Nutr Res., 11: '2. Zemel, M.B. (1988). Am J. Clin. Nutr., 48: (