§§§ WlHHIIIIIIIHHHIHWWWINllHllilWllH’Wll l 13!! U!!! I HI I!!! (l!!! M ll Hill ("I l! I! Ill! ”11 l L IB R A R Y 1293 231 007 Michigan State [Inivcrsh3r This is to certify that the thesis entitled Effect of Dietary Fiber on Mineral Metabolism in the Rat presented by Atossa Rahmanifar has been accepted towards fulfillment of the requirements for M.S. degree in Nutrition Major professor Date 11/14/77 0-7639 EFFECT OF DIETARY FIBER (N MINERAL METABOLISM IN THE RAT By Atossa Rahmanifar A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Hunan Nutrition 1977 ABSTRACT EFFECT OF DIETARY FIBER (N MINERAL METABOLISVI IN THE RAT By Atossa Rahmanifar The effect of dietary fiber on mineral metabolism was investigated in two groups of thirty male rats fed five different experimental diets for 4 or 8 weeks. The control diet was fiber-free. A wheat bran diet provided 3. 5% acid detergent fiber. Three diets containing purified fiber had 7.4% agar, guar gum or microcrystalline cellulose with pectin (MCC:P = 70:30). Fiber-fed animals had higher fecal weights and faster intestinal transit than controls and among them agar and bran-fed rats had the higher values. Animals fed fiber diets (except MIC-P) had higher fecal water holding capacity than controls. Body concentration and retention of calcium, iron and zinc were not influenced by purified fiber sources while copper values were higher compared to control. Bran-fed animals had higher intakes of all minerals but carcass analysis showed lower calciun and zinc and similar iron and copper concentrations compared to rats fed purified fibers . ACIG‘JGVIEDGHTENT S The author expresses her sincere appreciation to her major professor, Dr. Wanda L. Chenoweth for her guidance and constructive criticism through— out her graduate work and for her valuable help in the preparation of this manuscript. The author extends her acknowledgment and thanks to the members of her comnittee, Dr. Olaf Mickelsen for his counsel, Dr. Maurice R. Bennink for his helpful suggestions, and to Dr. Duane E. Ullrey for his help and allowing her to use the laboratory facilities. Special thanks are also given to Dr. John L. Gill for his helpful comments in the statistical analysis, to Dr. Pao K. Ku for his time and assistance in the mineral analysis, and to Dr. D. D. Makdani for his helpful suggestions. The author wishes to thank her husband, Kamyar, for his continuous support and help throughout her graduate work and her parents for their encouragement to pursue a higher degree of education. ii TABLE OF C(NTEN'I‘S ACIG‘JOWLEDGEMENI‘S . LIST OF TABLES INTROIIJCI‘IOI . REVIEW OF LITERATURE MATERIALS AND METHODS . Diets and experimental animals Mineral analysis . Rate of intestinal transit . Statistical analysis . RESULTS Food intake and weight gain . Fecal weights . Mineral utilization Calcium . . . . Iron . . . . Zinc . . Copper Effect of dietary fiber on intestinal transit DI SCUSSIQJ (DNCLUSIO‘J . . . REFERENCES . . . . . APPENDIX . . . . Standards for mineral analysis General reconmendations Artificial serum standards Primary standards. Working standards. Feed, feces and tissue standards. Primary standards. . Working standards . iii Page ii 13 13 17 18 19 20 20 20 20 27 31 31 38 49 57 57 57 61 61 62 Page Statistical evaluation . . . . . . 64 Oneaway analysis of variance . . 64 Two-way analysis of variance . . . 64 Bonferroni t-statistics . . . . 64 Experimental data.for individual animals 68 calciunl. . . . . . . . 68 Iron . . . . . . . . 69 Zinc . . . 70 Copper . . . . . . . . 71 Serun calcium concentration and henntocrit . 72 'iv LIST OF TABLES Table Page 1 Diet composition . . . . . . . . l4 2 Analyzed mineral content of the prepared diets . . l6 3 weight gain, fOOd intake and feed efficiency . . . 21 4 Fecal wet‘weight, dry weight and water holding capacity. . . . . . 22 5 Average daily'intake, fecal excretion and percent apparent absorption of calcium during three days of fecal collections . . . . 24 6 Tissue and serun calcium concentrations . . . . 25 7 Average daily intake, estimated body accumulation and percent retention (relative to intake) of calcium . 27 8 Average daily intake, fecal excretion and.percent apparent absorption of iron during three .days of fecal collections . . . . . 28 9 Average daily intake of iron and.hematocrit . . . 30 10 Tissue concentration, estimated body accumulation and percent retention (relative to intake) of iron . . 31 11 Average daily intake, fecal excretion and percent apparent absorption of zinc during three days of fecal collections . . . . . 32 12 Average daily intake, tissue concentration, estimated body accumulation.and.percent retention (relative to intake) of zinc . . . . . . . . 33 13 Average daily intake, fecal excretion and percent apparent absorption of copper during three days of fecal collections . . . 34 14 Average daily intake, tissue concentration, estimated body accumulation and percent retention (relative to intake) of copper . . . . . . . 36 Table Page 15 Percent excretion of polyethylene glycol after 16 and 32 hours of ingestion, Group B . . . . . 37 16 One-way analysis of variance . . . . . . . 66 17 TWO-way analysis of variance . . . . . . . 67 18 Percent apparent absorption, average daily intake, tissue concentration, total body accumulation and percent retention of minerals for individual animals. . . . 68 a. Calcium . . . . . . . . . . 68 b. Iron . . . . . . . . . . 69 c. Zinc . . . . . . . . . . 70 d. COpper . . . . . . . . . . 71 19 Serum calcium concentration and hematocrit . . . . 72 vi INTTDDUCI‘ICN Dietary fiber has been ignored by most human nutritionists and physicians probably because it is indigestible, contributes no calories and has negligible nutritional value. Dietary fiber has recently been suggested to have an important role in the gut and in decreasing the incidence of many diseases . Based on epidemiologic evidence, lack of fiber in the diet is suggested to be largely responsible for many diseases camnn in the Western world such as diverticular disease of the colon, colonic cancer, hiatus hernia, coronary heart disease, diabetes mellitus and obesity. Increase in fiber intake has been shown to decrease digestibility of energy sources. The chelating character of dietary fiber, its ability to decrease intestinal transit time and its water absorption capacity have focused attention on the question of whether fiber may alter mineral availability. Wheat bran has been used as a source of dietary fiber in nunerous studies and was shown to interfere with the availability of minerals in huran subjects and experinental aninnls. High intakes of whole wheat bread have been associated with occurrence of mineral deficiencies in Iranian villagers which has been partly attributed to the substantial anmmts of phytate in wheat bran. Effects of purified fiber sources on mineral metabolisn have not been studied extensively. The purpose of this research was to study the effect of different purified fiber sources and wheat bran on calcium, iron, zinc and copper metabolism REVIEW OF LITERATURE Dietary fiber has long been disregarded by most human nutritionists and medical workers as having an essential role in the gut and in the maintaining of man's health, probably because it contributes no calories and has little nutritional value. Energy intake, time for the passage of intestinal content through the alimentary tract, levels of intra- colonic pressures, numbers and types of colonic bacteria, as well as the levels of serum cholesterol and changes in bile salt metabolism have all been shown to be related to the amount of dietary fiber consumed (l) . A hypothesis based on epidemiological evidence and strongly promoted by Burkitt, Painter and others, is that the lack of dietary fiber is largely responsible for many diseases canton in and characteristic of modern Western civilization (1-3) , including diverticular disease of the colon (3,4), colonic cancer (2,5,6), appendicitis (1,6), gall bladder disease (1), hiatus hernia (1,7), deep vein thrombosis (l), coronary heart disease (8), ischemic heart disease (9), diabetes mellitus (9), and even obesity (10). In defining fiber, the distinction between dietary fiber and crude fiber will serve as a starting point. Dietary fiber is that part of plant material ingested in the diet which is resistant to digestion by the secretions of the human gastrointestinal tract ( 11-13). McCance and Lawrence (14) called this fraction of the diet "1mavailable carbohydrate", although it is not strictly unavailable or entirely carbohydrate. Dietary fiber consists of cellulose (a linear polymer of repeating 1-4 linked D—glucose mits), hemicelluloses (carplex polymers of xylose, arabinose, glucuronic acid, galactose, mannose and.occasionally other sugars), pectin (a polymer of galacturonic acid, arabinose and galactose), gums, mucilages and lignin (polymer of phenyl propane) (15,16). (bllulose is the most abundant component found in the cell wall of higher plants and lignin is the main non-carbohydrate carponent . True intakes of dietary fiber are very difficult to estimate and current food.composition tables do not report dietary fiber content of foods. Instead, crude fiber values are given which represent only a relative idea of the total fiber content. crude fiber, as defined.by the Association of Official Analytical Chemist (11) is the fibrous residue of a feeding material left after extraction with solvent, dilute aqueous acid and dilute alkali . However, the crude fiber method as an estimate of the fiber in foods has same defects illustrated by the fact that the acid and alkali sequential extraction may remove up to 80% of the hemicellulose, 50% to 90% of the lignin, and 20% to 50% of the cellulose (15) . There are other methods available for fiber determination in foods which can give'moreimeamingful research information than those available from the crude fiber determination. (he of these methods is the Southgate method (17,18) which differentiates between cellulose, hemicellulose, lignin and water soluble polysaccharides in human food- stuffs. Van Soest developed the detergent system of fiber analysis (19-21). The neutral detergentprocedure for cellawall constituents provides a rapid method for the total fiber determination in vegetable feedstuffs (20), and acid detergent method determines fibrous materials composed mainly of lignin, cellulose, and insoluble minerals (21). The difference between acid detergent and.neutral detergent fiber is an indication of hemicellulose. Spiller and Men (22) have recently recommended the classification of plant fibers into two subgroups: l) purified plant fibers in which structural polymers such as cellulose, hemicellulose and pectin have been isolated and purified, and 2) non-purified plant fiber in which the plant cell wall is in its natural state containing all associated substances such as trace minerals, guns, mucilages, glycoproteins. Therefore, it is important to consider that any effect attributed to non—purified plant fibers may be caused by associated components of the cell wall. The fibrous material in the human diet presumably represents the non-digest ible organic material . However, the bacteria found in the normal large intestine of humans are known to digest plant fibers by virtue of the production of "cellulases" (23). End products of the digestion are short chain or volatile fatty acids, water, carbon dioxide and methane. The first major investigation in man was by Williams and Olmsted (24), who fed various fibrous materials to humns and studied the feces to determine the relative disappearance of hemi— cellulose, cellulose, and lignin. The microflora of the large intestine were able to digest hemicelluloses to a significant extent, cellulose to a limited extent and left lignin unchanged. More recently, Southgate and Dumin (25) used natural foods, primarily whole mealbread, as a source of cellulose and slowed a range of mean cellulose digestibility from 15% for yomg men to 55% for elderly men. It has been known for many years that adding plant fibers in the diet will increase the fecal bulk and improve bowel habits. Cowgil and Anderson in 1932 fed 1 - 1% oz. of bran, either acid washed or unwashed, to five adults after a period on low fiber diet. The mean daily fecal weight increased from 114 g/day (during the low fiber period) to 193 g/day with bran in the diet (26). Significant increases in the wet weight of stool in eight subjects with bran or cellulose added to their diet have been reported recently (27). Burkitt, Walker and Painter (28) determined the amount of stools passed by various ethnic groups together with the speed of transit of food residues through their in- testinal tract. Some individuals required as long as 140 hours for the undigested particle to traverse the entire gastrointestinal tract and the weight of stool was about 100 g/day, while other individuals with stool weights around 400 to 550 g/day had shorter transit time (20—40 hours). They concluded that the more refined the diet is, the Staller the stool and the slower the passage of the food residues through the intestine. In contrast, high fiber diets produce stools which are soft, bulky, and traverse the gastrointestinal tract rapidly. This association was confirmed by the work of Payler M. (29), who found a decrease in transit time from 2.75 1- 1.6 to 2.0 1- 0.9 days upon the addition of bran (20 g/day) to a normal English diet. Laxative properties of bran have been known for many years (26,30,31), but deceleration of fast transit time was an unexpected finding in the study of Harvey and coworkers (32), suggesting that bran in sore way "normalizes" colonic behavior and low fiber diets induce abnormal colonic behavior either in the form of stasis or rapid transit. Eastwood M. (33) reported no change in transit time in 8 healthy subjects with bran or cellulose (16 g/day) added to their usual diet in spite of a signifi- cant. increase in stool weight . The mechanism by which dietary fiber affects fecal transit and bulk is not completely understood. Cummings (34) suggested that these changes 6 might be due to the microbial production of volatile fatty acids from the unabsorbed carbohydrates in the gut . The cathartic effect of volatile fatty acids may thus lead to decreased water absorption and increased fecal bulk. The mechanism producing increased fecal weight may be explained by the water absorbing properties of fiber which differ with various corponents of dietary fiber (35, 36). Cellulose has a moderate water holding capacity, pectic substances and hemicelluloses have a high capacity, and ligiin has a low water absorption capacity (36). Unabsorbed fiber also acts as a simple bulking agent to promote colonic peristalsis and faster transit (34). Siorter transit time caused by dietary fiber may result in reduced time available for digest ion and absorption of nutrients. Moran and Pace (37) reported that increased intakes of fiber as wheat fiber are accompanied by decreased digestibility of energy sources. This association wasconfirmed recently by the work of Southgate and Durnin (25). In- creasing the fiber intake resulted in a greater fecal loss of carbohydrates and in most instances of nitrogen and fat. The possible binding capability of dietary fiber to carbohydrates, lipid fractions and nitrogenous compounds, and its ability to shorten the transit time, have focused attention on the quest ion of whether fiber may alter mineral availability. Chelating properties of plant fibers, their water absorp- tion capacity (which dilutes the intestinal content) and the fast transit may provide mechanisms by which fiber could affect availability of mineral elements (16). Spiller and Amen (16) believe that a nutrient can possibly be rendered less available to such an extent that a new RDA (Recommended Daily Allowance) would have to be suggested. The mechanism of decreased availability may stem from the ionic character of mineral elements and chelating or complexing properties of plant fibers (38). Concentrat ion of the elements involved, concentration of the corplexing agent, pH, stability and absorbability of resultant corplex are all important factors which may affect complex formation and as a result mineral availability (38) . Some complexes yield their metal during digestion, others may resist digestion and prevent the metal from becoming available. Phytate is a complexing agent which occurs naturally in all food of plant seed origin and occasionally in roots, tubers and certain fruits (39). The proper chemical designation of naturally occurring phytic acid is myoinositol, l,2,3,4,5,6 - hexakis (dihydrogen phosphate) (40) . The anionic character of phytate makes it ideal for forming corplexes with mineral elements, and it has been shown to decrease the availability of calcium, iron, zinc, copper, manganese and magnesium (39) . Binding substantially decreases the effective metal concentration in the lumen leading to lowered rate of uptake by the mucosa. The acidic conditions of the stomach and partial digestion of protein result in partial dissociation and release of the phytate in protein which causes the dissociation of most cations from phytate (38) . As the pH increases in the pylorus and duodenum, the dissociated phytate begins to reform complexes with the elements which had been dissociated at the lower pH, however, these complexes are insoluble at the higher pH's. Phytate is not absorbed and, therefore, anything complexed to phytate will not be absorbed (38). The rachitogenic effect of phytate in cereals was originally identified by Bruce and Callow (41) under laboratory conditions. Phytate added to diets of rats (42, 43) has been shown to cause rickets by decreasing absorption of calcium from the intestine. Consumption of large amounts of high-extraction flour has been shown to reduce calcium absorption in human volunteers (44-47). McCance and Widdowson (44) reported reduced calcium absorption in human subjects taking high extraction wheat flourias 40—50% of their total caloric intakes. Negative balances of calcium were reported in two subjects during a 20—day period of high fiber consumption in the form of whole wheat bread which supplied 58.3% of the total energy (45) Increased fecal losses of metal ions including calcium explained the poor availablity and negative balance. A significant lowering effect on plasma calcium following consumption of 18-100 g wheat bran per day (median 38 g) for 4—9 weeks was attributed to interference with calcium absorption in human adults (48). Hydrolysis of phytic acid by the enzyme phytase to inositol and six phosphoric acid molecules is an important factor in controlling the effect of phytates on iron and calcium absorption (49). Phytase has been deronstrated in the gastrointestinal tract of albino rats, guinea pigs, rabbits, pullets, chicks, and ruminants (49), but its pre- sence in huran digestive tract is still open to question. Contrary to the belief that persisted for many years, Bitor and Reinhold (50) claimed the existance of phytate-splitting esterases in man. I-bwever, destruc- tion of phytate in the gut apparently is not sufficient to overcome the effects of high phytate intakes (47). Iron absorption depends on many coiplex and interrelated factors, the most important ones being body iron stores and the degree of erythropoiesis (51) . These factors and the regulatory effect of the intestinal mucosa on iron uptake result in iron deficient rats absorbing more iron than normal rats (52, 53). According to Moore (54), only 5 to 10 percent of food iron is absorbed in normal adults, which may fall below 3 percent in diets with a low calcium, high phosphorous, and high phyt ic acid content (49) . Ingest ion of brown bread by human subjects resulted in lower iron absorption than when a white bread diet was consumed, in spite of the higher iron content of the former diet (55). Similar experiments have confirmed the inhibitory effect of whole wheat bread on iron absorption which appears to be mainly due to its phytate content (56-59). A significant decrease in iron absorption from breads containing equal to or greater than 3.3% bran has been reported (58). Contrary to these findings, Walker and coworkers (60), reported no effect on iron retention with consumption of brovm bread in human subjects. A relationship between bulk of dietary intake and iron absorption has been reported, suggesting the inhibitory effect of high bulk diets on iron absorption (61). A significant decrease in serum iron of elderly patients taking 10 to 20 grams of wheat bran daily in their diet for six weeks recently was demonstrated, indicating a possible inhibitory effect of bran on iron absorption (62) . O'Dell and Savage (63) were the first workers to suggest that the difference in the availability of zinc between animal and plant proteins might be due to the phytic acid content of the latter. They reported that phytate added to a casein-gelatin semipurified diet decreased growth in chicks similar to that seen with a soy protein diet (63). This observation has since been confirmed for chicks (64-66) and has also been shown for pigs (67) and rats (68, 69). A mechanism proposed by (berleas __e_t_:a_l_. (68, 69) involved the formation of highly insoluble salts of phytate in the upper segments of intestine. This effect of phytate is augrented by dietary calcium, forming a Zn-Ca—phytate complex that reaches its maximum insolubility at pH of about 6, the approximate 10 pH of the duodenum and upper jejunum in which zinc and many other mineral elements are absorbed (70). The results of studies of zinc absorption from ligated loop of rat duodenum in ALtu' have shown marked reduction of mucosal uptake, binding and absorption of 65Zn when phytate was present (71). Fecal zinc balance and whole body analysis of young rats showed an increase in fecal zinc excretion and reduced whole body retention of zinc when 10 g/kg phytate was added to both zinc supplemented (15 mg/kg) and zinc-deficient (0.5 mg/kg) diets (71). Recent studies in rats have indicated that zinc is continually secreted and reabsorbed across the intestinal mucosa (72—74) . A possible explanation is that phytate may affect overall zinc utilization both by affecting the reabsorpt ion of endogenously secreted zinc and by direct action on the absorption of dietary zinc (71) . Another alternative explanation for these results is that presence of phytate, a strong zinc carplexing agent in the lumen of the small intestine, may extract zinc either from the intestinal mucosal cells or across the mucosal tissue fram the blood and tissues ( 71). Impaired zinc retention has also been reported in human subjects maintained on phytate rich diets (46). In addition to its effect on zinc, iron and calcium, dietary phytate has also been shown to reduce the availability of copper ( 71), magnesium (45, 75, 76) and manganese (71). Several findings have led to quest ions as to whether phytate is the only or the most important complexing agent in whole wheat flour. Phytate consumed in purified form by human subjects was less effective in causing negative balances of zinc and calcium than were the equivalent amounts in the form of wholemeal bread (46). Contrary to this report, destruc- tion of phytate of wholereals by yeast during bread—making increased 11 zinc solubility and its uptake by rat intestine (77). Reinhold M. (78) reported that removal of phytate from wholereal or bran by acid extraction or by action of phytase increased the affinity for binding metals in vii/L0, and concluded that it is fiber which is mainly responsible for the interference with dietary metal absorption by the intestine and phytate would be important only to the extent that it escapes digestion. Cellulose, a purified plant fiber has been shown to interfere with the absorption of zinc and iron (79, 80). Addition of 10 grams of cellulose to the daily diets of low and high fiber content resulted in increased fecal excretion, negative balance, and reduced serum concentration of calcium and zinc in three hulan subjects after 20 days (81) . Cellulose phosphate is known to decrease calcium absorp- tion and is used to control hypercalcemia (82). The effect of different levels of dietary fibers on calcium, iron and copper absorption was studied in male rats during an 18-day period (83). The dietary fiber sources included peanut hulls, (em pericarp and isolated cellulose. These fiber sources increased calcium absorption and decreased iron and copper availability irrespective of source or level of fiber. Pectin ingested as 5 or 10 percent of the diet, reduced food intake and calcium absorption in weanling rats (84). Branch e331; . (85) suggested that the reduced calcium availability caused by intake of certain sources-.of dietary fiber may be related to the ionized carboxyl groups of uronic acids which can bind calcium and those fibers with higher methyl—substituted uronic acids are unavailable for calcium binding. Dietary phytate and oxalate were suggested to increase calcium binding. In sumary, it has been known for many years that cereal grains interfere with the absorption of bivalant ions due to their high content 12 of phytate. Impaired growth due to zinc deficiency has been shown in both animals and huran subjects maintained on phytate-rich diets. Special interest has centered upon the effects of wholemeal bread because of its importance in the diets of many populations and the disturbances in mineral metabolism associated with its consumption. Phytate when ingested in large amounts interfered with availability of calcium, iron, zinc, copper, magnesium, and other minerals both in hurans and animals. Dietary fibers other than bran also have been shown to reduce mineral availability but have not been studied as extensively. MATERIALS AND METHODS Diets and emerimental animals . The experiment was conducted to study the effect of dietary fiber on the metabolism of calcium, iron, zinc and copper. Sixty male, Sprague- Dawely rats weighing approximately 200 g were divided into two groups of thirty rats, Group A and Group B. Group A were maintained on their respec- tive diets for 4 weeks and Group B for 8 weeks. Each group was divided into five subgroups of 6 rats each and fed five different diets. The control diet was fiber-free. Three diets had 7.4% purified fiber from one of the following sources: microcrystalline cellulose-pectin (MCC-P), agar, and guar gun. A fifth diet containing wheat bran provided 3.5% acid detergent fiber (15.8% neutral detergent fiber). Table 1 describes the composition of the experimental diets. All diets were calculated to have the same protein-calorie ratio. Except for the presence and source of fiber, the control, MCC-P, agar and guar gum diets had the same amourt of ingredients (per 481 kcal) . The bran diet was different because bran itself is a source of protein and carbohy- drate1 , and other ingredients had to be adjusted to provide the same total amount of protein and calories. A mineral mixture was used to provide the requirement levels of calcium, iron, zinc and copper for rats, using the values recommended by the National Research Council, 1972 (86): calcium, 116% protein, 61.9% total carbohydrate, 11.9% fiber and 4.6% fat (Composi- tion of foods, U.S.D.A. Handbook, No. 8, 1963) 13 14 Table 1. Diet composition (g/481 kcal) Ingredients Control iMCC-P1 Agar2 Guar gum3 Bran? Caseins 12.3 12.3 12.3 12.3 9.2 White flour6 60 60 60 60 40.2 Fat’ 22 22 22 22 22 Purified fiber - 8 8 3 - Wheat bran - - - - 31 Cholesterol 1.0 1.0 1.0 1.0 1.0 Choline 0.2 0.2 0.2 0.2 0.2 DL-methionine 0.3 0.3 0.3 0.3 0.3 Vitamin mix‘ 1.0 1.0 1.0 1.0 1.0 Mineral mix"‘° 3.2 3.2 3.2 3.2 3.2 1Microcrystalline cellulose/pectin (70/30) , IMC Corporation, Marcus Hook, Pennsylvania. 2Bacto-Agar, Difco Laboratories, Detroit, Mfichigan. 3Guar gum, F-G-70-70, Hercules, Inc., Wilmington, Delaware. |'Lucky Table Wheat Bran (for human consumption) , International Multifoods, Inc. , Minneapolis, Minnesota. 5Sodium Caseinate, western Dairy Products, Charlotte, North Carolina. “Enriched bread flour, Washburn Special, General Mills Inc. , 9200 Wayzata Boulevard, Minneapolis, Minnesota. 710.5 g corn oil (Mazola) + 11.5 g lard (Farmer Peet's Shortenin' - pork and.beef fat - Peet Packing Company, Chesaning, Michigan) to provide p s - 1. ”Camposed of (in mg/kg diet): thiamin HCL, 22; pyridoxine, 22; riboflavin, 22; Ca pantothenate, 663 P-amino benzoic acid, 110; menadione, 50; inositol, 100; ascorbic acid, 200; niacin, 100; Vitamin.Blz, 0.03; biotin, 0.6; folic acid, 4; In IU/kg diet - Vitamin A acetate, 20,000; alpha toc0pherol, 97.7; cholecalciferol, 220. 9Composed of (g/100 g mineral mix): Cams, 20.0; CaHPOu, 28.3; NazHPOu, 20.3; K01, 22.8; MgSOu, 7.2; MnSOu - H20, 0.5; 011501.. 51120, 0.06; 10FeSO., . 7H20, 0.54; ZnSO. . 7H20, 0.2; K103, 0.003; NaSeOs, 0.0003. Ca;P = 1.4 15 5000 ppm; iron, 35 ppm; zinc, 12 ppm; and c0pper, 5 ppm. For the other minerals the amounts used were similar to the amounts usually added to experimental diets from commercial mineral mixes . Because of variation in the amounts of minerals provided by dietary constituents other than the mineral mix, the total mineral content of the diets was not the same (Table 2) . Higher levels of dietary iron occurred because enriched white flour was used. Animals were housed individually in hanging wire mesh, stainless steel cages and given food and deionized distilled water ad libitun. Water was given in bottles with plastic caps and stainless steel tubing. Constant temperature and humidity conditions and a lZ-hour light-dark cycle were maintained. After a 3-day adjustment period in the cages all rats were transferred from a commercial rat pellet1 to the control diet and prefed for two weeks in order to adapt all animals to the desired and equal levels of minerals . Food intake and weight gain were determined weekly. Group A: animals were fed for 4 weeks. At the end of the third week, three 24-hour fecal collections were made to measure fecal wet weight, dry weight and mineral content. At the end of 4 weeks, animals except those fed guar gum were anesthetized with ether and blood was drawn from the heart. Rats were then killed, stomach and intestinal contents removed and carcasses frozen for later analysis of minerals . Hematocrit was determined using dry heparinized capillary tubes which were filled with blood, centrifuged2 and then read. Clotted blood samples were centrifuged for 15 mimltes at 1500 g3, serum removed and frozen for calcium analysis. 1Lab Blox, Wayne Laboratory Animal Diets, Allied Mills, Inc., Chicago, Illinois 60606. 2Micro-Capillary Centrifuge, Model MB, International Equipment Company, Needham, Massachusetts. 3International Centrifuge, Model UV, International Equipment Company, Needham, Massachusetts. 16 Table 2. Analyzed mineral content of the pmpared diets. Diet Ca Fe Zn Cu PP‘“ Control 5302 67.9 21.1 4.8 MIC-P 4894 60.1 17.5 4.3 Agar 4984 56.6 20.9 5.3 Guar gun 4710 54.3 21.6 4.5 Bran 4856 92.9 46.0 8.4 17 Rats fed the guar gum diet were killed one week later because they had been started on diet one week later than the other animals. Group B: animals were fed for 8 weeks. After 7 weeks, feces were collected for 72 hours for analysis of mineral content. After completion of fecal collections rats were tube-fed with approximately 2 m1 radioactive 1“(l-polyethylene glycoll. (PEG). Dosage of radioactive material administered to each animal was determined by total counts recovered in the feces. Feces were collected after 8, 16, 32, 40, 56, 64 and 80 hours of PEG intubation and frozen for later estimation of rate of intestinal transit. At the end of 8 weeks, animals were killed and blood and carcass prepared and frozen for later mineral analysis using the same procedure as for Group A. Mineral analyses . All glassware used for preparation and analysis of samples were acid washed. Wet feces collected in each 24-hour period were weighed and then oven-dried at 60°C to bring to a constant dry weight. Dried feces collected in 3 days were ground in a Wiley mill”. Duplicate aliquots of ground feces (500 mg) and diet (2 g) were weighed into 250 ml Phillips beakers to be wet ashed using 30 ml concentrated HNO3 and HCIlO3 (1:1) . Digestion was con- tinued urtil volume was reduced to l to 2 ml. After the samples had cooled, they were diluted to 50 ml using deionized distilled water. For calcium analysis, a second dilution (1:10) with distilled deionized water was necessary. The standards contained a mixture of minerals in Specific concentrations which were prepared from a mixture of primary standards (Appendix, p. 61). Diluted samples for calcium analysis and working 110 mg of PEG = 1 11C = 2.2 x 106 dpm; Amersham/Searle Corporation, Arlingtm Heights, Illinois 2Arthur H. Thomas Co., Philadelphia, Pennsylvania 18 standards were further diluted (1:20) with 10,000 ppm strontium chloride1 and analyzed for calcium. For iron and zinc analysis, a second dilution (1:10) of ashed samples with distilled deionized water was also needed; a second dilution was not necessary for capper analysis. Prepared samples were then analyzed by atomic absorption spectmphotometryz. The carcasses were thawed at room temperature, autoclaved3 for 60 minutes in glass bottles at 112°C, weighed, and homogenized with 400 m1 deionized distilled water in a stainless steel Waring blender“ and approximately 150 ml of homogenate were frozen. Duplicate aliquots (10 g) of thawed carcass homogenate were digested by wet ash procechn‘e and prepared for calcium, iron, zinc, and copper analysis. Procedures and dilutions used were the same as those described for feed and feces. Duplicate aliquots(l ml) of thawed serum were deproteinized with 4 ml of 12.5% trichloroacetic acid. The supernatant was diluted (1:2) with strmltium solution (20,000 ppm). Serum calcium concentration was determined by atomic absorption spectrophotometry using standards containing a mixture of minerals in concentrations similar to those fourd in serum (Appendix, p. 58) . Rate of intestinal transit. Feces collected after PEG intubation were thawed at room temperature, weighed, transferred to small beakers and diluted to 5 ml by addition of distilled water. After approximately 15 minutes, fecal samples and water were mixedthoroughly using a Virtis hamogenizers. Duplicate aliquots (100 ml) 130.5 g SrClz.6H20 + S 3 NaCl per liter of deionized distilled water. Reagents obtained from J. T. Baker Chemical Co. , Phillipsburg, N.J. 2Model 453, Instrulentation Laboratories, Inc. Lexington, M.A. 3American Sterilizer Company, Erie, P'.A. I’Waring Blender, Model-CB-4, Waring Products Company, New York, N.Y. 5Model 23, The Virtis Company, Gardiner, N.Y. 19 of the homogenate were mixed with 5 ml of scintillation cocktail1 and radioactivity determined by liquid scintillation. Percent excretion of PEG in each collection was calculated by comparing its radioactivity with the total Immber of counts excreted in all feces. Statistical allaysis . Data were analyzed for statistical significance at p < 0.05 using analysis of variance and the Bonferroni t-statistics for comparison between groups (87) (Appendix, p. 64). Three sets of comparisons were made: 1) Control vs. NBC-P, Agar, Guar gun and Bran combined. 2) Bran vs. MCC-P, Agar and Guar gum combined. 3) Control. vs. MIC-P, Agar and Guar gum combined. 11 liter of scintillation cocktail contains: 333 ml triton x-lOO, 667 ml toluene, 4 g ppo (2,5-diphenyloxazole) and 200 mg dimethyl popop (1 .4-bis [2- (4-Methyl- 5-phenyloxazoly1)]-benzene) . RESULTS Food intake and weight gain. Total weight gains for the animals in Groups A and B did not appear to be influenced by any of the fiber sources. However, analysis of variance indicated that food intake was increased with fiber -supplemented diets and among them, the highest food intakes were shown in bran-fed rats. Therefore, fiber-supplemented diets appeared to have lower feed efficiencies with bran being the lowest (Table 3). Fecal weights . Fecal weights and water holding capacity were also influenced by fiber containing diets (Table 4). All fiber sources increased the fecal wet and dry weights with the bran grotp having the highest weight. Fecal water holding capacity was higher with agar, guar gum and bran while MIC-P diet had lower water holding capacity than the fiber- free diet. Mineral utilization. Calcium. The data presented in Table 5 indicate that animals fed bran had significantly higher daily fecal calcium output during the 3 days of fecal collection than other fiber containing diets which resulted in 20 21 . Table 3. Weight gain, food intake and feed efficiency. Diets Total wt. gain1 Total food intake1 Food efficiency1 3 g g wt. gain/g food intake Group A Control 168. 5 4582 0 . 37" ICC-P 161 . 3 473 0 . 34 Agar 169.5 491 0.34 Bran 167.8 5303 0.32 Group B Control 259. 4 9362 0 . 28 MIC-P 253.3 973 0.26 Agar 272.4 1075 0.25 Guar gum 256.2 954 0.27 Bran 270. 2 1131 3 0. 24 SB! . 10.64 19.00, 0.01 1Means for six rats. 2Significantly lower (P < 0.05) than fiber-supplemented diets combined. ’Significantly higher (P < 0.05) than purified fiber diets combined. “Significantly higher (P < 0.05) than fiber-supplemented diets combined. 22 Table 4. Fecal wet weight, dry weight and water holding capacity (WI-1C). 1 Group A Diets Fecal wet weights2 Fecal dry weights2 M243 g/day g/day 4 Control 0.87“ 0.69“ 24.4“ MCII-P 1.84 1.58 15.4 Agar 2.71 1.75 54.8 Guar gum 1.92 1.28 48.4 Bran 4.425 3.205 37.6 SEM . , . . 0.171 0.081 4.36 :Fecal collection made after 3 weeks on diet. 4322’; £355; Egan}, = fecal wet weight - fecal dry weight fecal dry weight x 100 “Significantly lower (P < 0.05) than fiber-srpplemented diets combined. 5Significantly higher (P < 0.01) than purified fiber diets combined. 23 significantly lower apparent absorption1 of calcium in those animals. FCC-P, agar, and guar gum diets did not influence fecal excretion and apparent absorption of calcium compared to controls. Total carcass analysis (Table 6) revealed a lower tissue concentration of calcium in animals maintained on fiber-supplemented diets although values reached statistical significance only in Group B. The lowest tissue cal- cium concentrations were fould in the bran-fed rats . Carcass values for calcium and other minerals in Group A animals fed guar gum diet are not reported because these animals were killed one week. later -than other animals. Serum calcium concentrations were also lowered by the bran diet compared to other fiber supplemented diets, both in Group A and Group B. Serum calcium concentrations in animals supplemented with MCC-P and agar were higher than controls in Group A, but this difference was not seen in Group B. The estimated whole body content of calcium and other minerals at the beginning and end of the dietary treatment was used to calculate the total and percent body accumulation and retention of minerals. In order to have an estimate of whole body mineral content at day zero, the average tissue mineral concentration of animals maintained on control diet for 28 days was used as a standard or constant. Concentrations at day 28 had to be used because animals had not been killed at day zero. A standard tissue mineral concentration was used based on the fact that all animals were on the same commercial diet before starting the experiment, pre-fed the control diet for two weeks, and therefore assumed to have similar tissue concentration of minerals. For all animals, the initial body weights were 1 ' ' t - Fecal calcium Apparent absorption = Calcrum m ake Calcium intake 24 Table 5. Average daily intake, fecal excretion and percent apparent absorption (of calcium during three days of fecal collections} Diets Intake2 Fecal excretion2 Apparent absorption2 Ins/day mg/day Group A Control 86.6 33.7 61.2 Mai-P 79.9 30.7 61.8 Agar 88.6 36.1 60.5 Guar gum 77.5 35.7 51.7 Bran 92.53 48.8“ 47.65 Group B Control 86. 0 47 . 6 . 43. S MIC-P 84.8 48.4 42.8 Agar 98.0 55.7 43.2 Guar gum 79.8 46.5 40.8 Bran 99.03 67.2“ 32.25 $341 3.46 2.60 1.99 1Fecal collection made after 3 weeks in Group A and 7 weeks in Group B. 2Means for six rats. ‘ 3Significantly higher (P < 0.05) than purified fiber diets combined. “Significantly higher (P < 0.01) than purified fiber dietscombined. 5Significantly lower (P < 0.01) than purified fiber diets combined. 25 Table6. Tissue and serum calcium concentrations . Diet Tissue concentration‘ Serum concentration‘ mg/g body wt. mg/100 ml Group A Control 5.3 9.62 Mal-P 5.1 11.7 Agar 5.2 11.4 Bran 4.8 9.83 Group B Control 6.1“ 10.1 MOS-P 5.6 11.4 Agar 5.6 10.3 Guar gum 5.6 10.1 Bran 4.83 8.73 834 0.27 0.52 1Means for six rats. :Significantly lower (P < 0. 05) than MCC- P and agar diets combined. :Significantly lower (P < 0. 05) than purified fiber diets combined. “Significantly higher (P < 0. 05) than fiber- -supplemented diets combined. 26 multiplied by this standard to get an estimate of body mineral content at day zero. Differences between the estimated body content of minerals at the beginning and end of the dietary treatment (total body accumulation) then was used to calculate the percent body accumulation of minerals during the study period: % body mineral accumulation = Final body mineral - Estimated initial body mineral x 100 Final body mineral When differences in dietary intake of minerals were taken into accoult , results were expressed as percent mineral retention relative to intake: Whole body mineral retention relative to intake (%) = Accumlated mineral in body (Final-initial) x 100 Total mineral intake None of the purified fiber diets appeared to affect body accumulation of calcium (Table 7). Bran-fed animals showed lower body accumulation of calcium while they had significantly higher average daily intake of calcium compared to other fiber fed animals. As shown in Table 7, the bran diet Isignificantly reduced the proportion of the dietary intake of calcium which was retained, while purified fiber diets had no effect on percent retention of calcium. Iron. The average daily intake , fecal excretion and percent apparent absorption of iron in Groups A and B during three days of fecal collection are shown in Table 8. Daily intake of iron was considerably higher in animals with bran in their diet which resulted in higher fecal excretion of iron compared to animals on purified fiber diets. Animals fed purified fiber diets showed similar intake and fecal excretion of iron to those on control diet. Variations in the amoult of iron intake and excretion in feces between different animals resulted in wide ranges of positive and 27 Table 7. Average daily intake, estimated body accumulation and percent retention (relative to intake) of calcium. 1 Diets Intake2 ’ 3 Accumulation’ Retention3 mg/day Total (mg) % % Group A Control 82 828 42.9 34.0 MIC-P 80 741 38 . 8 31. 4 Agar 84 896 44.7 37.0 Bran 89“ 625 35.4 24.45 Group B Control 87 1648 59 . 5 33. 0 MIC-P 85 1319 53. 4 28. 1 Agar 96 1460 57 . 0 27. 2 Guar gum 81 1349 54.9 29.6 Bran 98“ 10785 50.0 19.75 SEM 2.24 104.5 2.91 _ 3.17 1See text for explanation of calputation of body accumulation and retention. 2[hiring the entire period of 4 or 8 weeks. S’Means for six rats. I'Significantly higher (P < 0.05) than purified fiber diets combined. sSignificantly lower (P 2 0.05) than purified fiber diets combined. 28 Table 8. Average daily intake, fecal excretion and percent apparent absorption of iron during three days of fecal collections. Diet Intake2 Fecal excretion2 Range of ug/day u g/day apparent absorption2 % Group A Control 1110 774 20.7 to 37.8 MCC-P 982 723 18.2 to 34.4 Agar 1007 . 708 25.7 to 35.3 Guar gum 893 826 -4.2 to 23.9 Bran 17703 15023 5.4 to 21.2 Group B Cmtrol 1102 924 -4.4 to 28.5 MIC-P 1042 911 -1.1 to 19.4 Agar 1114 968 7.1 to 18.5 Guar gum 920 898 -Zl.5 to 16.6 Bran 18953 1872’ -6.0 to 10.9 saw 47 50 -" lFecal collection made after 3 weeks in Group A and 7 weeks in Group B. 2Means for six rats. 3Significantly higher (P < O. 01) than purified fiber diets combined. “Statistical analyses were not done because of wide ranges of negative and positive values within each dietary treatment. 29 negative apparent absorption within each dietary treatment; therefore, statistical analyses were not done on these data. Despite differences in the dietary level of iron, all animals in Groups A and B had similar hematocrit values , tissue concentration and percent body accumulation of iron (Table 9, 10). Bran-fed animals had significantly lower iron retention relative to their high intake in both Groups A and B while there was no significant difference between controls and animals fed purified fiber diets. Zinc. Wheat bran is also a rich source of zinc and when added to the basal diet resulted in higher dietary concentration and intake of zinc. Bran- fed rats showed considerably higher intake and fecal excretion of zinc during the three days of fecal collection while there was no signifi- cant difference between other fiber-fed rats and controls (Table 11). Values for percent apparent absorption of zinc during this period show a wide range of results from negative to positive within each dietary treatment. Carcass analysis showed a significantly lower tissue concentration and body accumllation of zinc in Group B animals maintained on bran diet than those on purified fiber diets, but this difference was not seen in Group A (Table 12) . Animals fed bran had significantly lower percent retention of zinc in the body relative to intake compared to animals on purified fiber diets. Animals supplemented with purified fiber sources showed no signi- ficant difference in tissue concentration, body accumulation, and percent retention relative to intake of zinc when compared to controls (Table. 12) . Capper. Table 13 shows the average daily intake, fecal excretion and percent apparent absorption of copper in Groups A and B after three days of fecal collection. Animals fed bran were exposed to approximately doubled 30 Table 9. Average daily intake of iron and hematocrit. Diets Intake1 2 z Homatocrit2 148/ day ‘5 Group A Control 10733 41.3 NBC-P 981 46.9 Agar 958 39.9 Bran 1700“ 46.5 Group B Control 1135 46.9 MIC-P 1044 48.2 Agar 1087 48.1 Guar gum 954 48.3 Bran 1877“ 46.8 SFM 30 1.201 1During the entire period of 4 or 8 weeks. 2Means for six rats. :Significantly higher (P < 0. 05) than MIC- P and agar combined. “Significantly higher (P < 0. 01) than purified fiber diets corbined. 31 Table 10. Tissue concentration, estimated body accumulation and percent retention (relative to intake) of iron. ‘ Diets Tissue concentration2 Accumulation2 Retention2 ug/g body wt. 'I'dtal (“181 Y % Group A Control 28.1 4.32 42.5 13.9 ICC-P 29.8 4.75 44.4 16.8 Agar 28.7 4.44 43.1 16.0 Bran 28.1 4.18 41.6 8.53 Group B Control 29.6 7.30 55.3 11.5 DEC-P 24.9 5.07 46.2 8.7 Agar 29.0 7.50 56.2 12.3 Guar gum 27.9 6.29 51.0 11.7 Bran 29.5 7.44 56.5 7.13 $34 1.202 0.401 1.94 1.13 1See text for explanation of computation of body accumulation and retention. 2Means for six rats. 3Sig1ificantly lower (P < 0.01) than purified fiber diets combined. 32 Table 11. Average daily intake, fecal excretion and percent apparent absorption of zinc during three days of fecal collections. Diets Intake2 Fecal excretion2 Range of ug/day ug/day apparent absorption2 % Group A Control 344 268 16.4 - 29.5 MIC-P 286 292 -48.4 - 21.1 Agar 372 263 16.9 - 34.0 Guar gum 355 279 -1.8 - 34.5 Bran 8763 8643 -6.5 - 7.1 Group B Control 342 343 -22.0 - 11.4 MCC-P 304 334 -24.1 - 0.8 Agar 411 383 -5.3 - 15.3 Guar gum 366 338 -35.9 - 17.7 Bran 9373 10853 -22.0 - -0.3 SEM 17.62 26.5 -" 1Fecal collection made after 3 weeks in Group A and 7 weeks in Group B. 2Means for six rats. 3Significantly higher (P < 0. 01) than purified fiber diets combined. “Statistical analyses were not done because of wide ranges of negative and positive values within each dietary treatment. 33 Table 12. Average daily intake, tissue concentration, estimated body accurllllation, and percent retention (relative to intake) of zinc. Diets Intake2 ’ Tissue cmcentration’ Accumlation’ Retention’ org/day ug/g body wt. Total Ting? 1 % Group A Control 333 19 . 3 2 . 99 42 . 7 31 . 0 MIC-P 286 20.0 3.12 43.2 37.3 Agar 354 20.2 3.25 44.2 31.7 Bran 841“ 19.4 2.90 42.0 11.95 Group B Control 353 26.1 7.66 65.4 38.6 MIC-P 304 24.6 6.21 60.5 36.8 Agar 401 25.3 7.64 65.7 33.9 Guar gum 367 27.2 7.80 65.8 38.0 Bran 929“ 22.15 5.935 59.3 11.45 SEW 11.21 0.33 0.324 2.01 2.17 1See text for explanation of computation of body accumllation and retention. 2During the entire period of 4 or 8 weeks. 3Means for six rats. “Significantly higher (P < 0.01) than purified fiber diets combined. 5Significantly lower (P < 0.01) than purified fiber diets combined. 34 Table 13. Average daily intake , fecal excretion and percent apparent absorption of copper during three days of fecal collections. ‘ Diets Intake2 Fecal excretion2 Range of uslday Halday apparent gbsorptimz Group A Coltrol 78.1 90.3 -52 - 6.0 Mic-P 70.4 91.3 -37.7 - -19.6 Agar 94.2 89.3 -19.2 - 28.5 Guar gum 74.3 98.2 -54.8 - ~12.6 Bran 160.83 169.43 -17.1 - 3.6 Group B Control 77.5 111.1 -76.6 - -24.l Mat-P 74.7 102.0 -52.7 - -22.6 Agar 104.2 116.0 -30.6 - 8.6 Guar gum 76.6 106.4 -109 - -6.8 Bran 172.13 196.89 -31.l - 0.6 SEM 3.51 6.91 - 1Fecal collection made after 3 weeks in Group A and 7 weeks in Group B. 2Means for six rats. 3Significantly higher (P < 0.01) than purified fiber diets combined. “Statistical analyses were not done because of wide ranges of negative and positive values within each dietary treatment. 35 intakes of capper compared to others because wheat bran is also a rich source of capper. The higher intake of copper was accompanied by a higher fecal excretion of copper in bran-fed animals compared to others with fiber in their diet. There was no sigrnificant difference between dietary intake and fecal excretion of copper in animals fed purified fibers and the fiber-free diet. Ranges of apparent absorption of copper in eadl dietary treatment also are shown in Table 13. Total carcass analysis revealed a lower tissue concentration and body accumlation of copper in control animals compared to those on fiber diets and this difference reached to a 99% significance level in Group B (Table 14) . Despite the markedly higher intake of copper in bran-fed animals, there was no significant difference in tissue concentration and body accumulation of copper compared to animals on purified fiber diets. This effect is further shown in percent retention of capper in body relative to the amount of intake which is significantly lower in bran-fed rats compared to those fed purified fiber diets . Effect of dietary fiber on intestinal transit. Percent excretion of radioactive polyethylene glycol after 16 and 32 hours of ingestion is shown in Table 15. Control rats excreted the smallest percentage of PEG colpared to others and among fiber containing diets , bran and agar- fed rats excreted the largest percent PEG . These results indicate that control diet produced the slowest rate of passage of intestinal contents whereas agar and bran supplemented diets produced the most rapid rate of transit. 36 Table 14. Average daily intake, tissue concentration, estimated body 'achnnlation, and percent retention (relative to intake) of capper.l Diets Intake’ ' 3 Tissue concentration’ ‘ AceumulatiOns Retention3 ng/day mg/g body wt. Total (mg) 7 % Group A Control 75.9 1.37 214 42.8 9.7 Mai-P 70.2 1.47 239 45.4 11.7 Agar 89.7 1.53 266 47.9 10.2 Bran 153.6“ 1.48 253 47.2 5.35 Group B Control 80.2 1.36‘5 3525‘5 53.06 7.2 mC-P 74.7 1.40 329 53.7 7.9 Agar 101.8 1.61 454 61.5 8.0 Guar gum 77.3 1.61 414 59.1 9.6 Bran 169.7“ 1.57 422 60.3 4.45 SEM 2.402 0.046 20.4 1.68 0.53 1See text for explanation of amputation of body accumlation and retention. 2During the entire period of 4 or 8 weeks. 3Means for six rats. “Significantly higher (P < 0.01) than purified fiber diets canbined. sSignificantly lower (P < 0.01) than purified fiber diets combined. ‘Significantly lower (P < 0.01) than fiber-swplemented diets canbined. 37 Table 15. Percent excretion of polyethylene glycol after 16 and 32 hours of ingestion, Group B. Diets % PEG excreted1 16 Hrs 32 Hr? Control 55 672 mC-P 58 77 Agar 79 95 Guar gum 54 76 Bran 71 963 SE! 6.66 3.83 1Means for six rats. aSignificantly lower (P < 0.01) than fiber-supplemented diets combined. ’Significantly higher (P < 0.05) than purified fiber diets combined. DISCDSSICN In this experiment, dietary fiber did not influence the growth rate. However, total food intakes were greater in animals maintained on fiber- supplannented diets and among them bran-fed animals had the highest intake. This higher intake may be explained by the lower caloric density or reduced energy availability of fiber-swplemented diets. However, animals adjusted their food intake and achieved similar weight gains to those fed fiber- free diet. It has been known for many years that adding plant fiber to the diet will increase fecal bulk. Increased daily fecal weights in human subjects with bran added to their diet have been reported by numerous investigators (24, 26, 27, 33). In the present experiment, fiber-supplanted diets increased fecal wet and dry weights and among them the wheat bran diet had the highest values. It is generally believed that fiber increases fecal bulk by virtue of its water retaining properties (34, 3S) . Animals fed agar, guar gun and wheat bran showed higher fecal water holding capacity 4 than those fed MCC-P and control diets. Fecal water holding capacity was greatest in rats fed agar; however, bran-fed rats had the highest fecal weights. Animals on MCIJ-P diet had lower fecal water holding capacity canpared to those on fiber-free diet although the former had higher fecal wet and dry weights. Eastwood (36) has shown that dietary fiber found in different fruits and vegetables varies in its water holding capacity. Dietary fiber itself adds to the fecal bulk because it is partially or completely non-digestible. Higher fecal dry weights in fiber-fed animals 38 39 may have bean partly due to this effect. Wheat bran added to the diet appeared to affect calcium utilization while purified fiber sources did not. Bran-fed rats had increased fecal Lbsses and decreased apparant absorption of calcium. Similar results were reported in human subjects consuming high amounts of whole wheat bread (45) . In the present experiment, values for apparent absorption of calcium (43 to 66% in Group A and 28 to 55% in Group B) are high compared to the results reported by Hansard and Plumlee (88) . These investigators found 37% apparant absorption of calcium in rats weighing 200 g andconsuming 102 mg calcium per day. Reduced serum and tissue concantrations , body accumulation and percent retention of calcium in bran-fed animals provided added evidence for the reduced calcium availability, despite the higher calcium intake of these rats canpared to other .animals. High intakes of wheat bran also have bean shown to reduce plasma calcium level in human adults (48) . Increased fecal losses and reduced availability of calcium have bean traditionally ascribed to the phytates present in wheat bran. The anionic character of phytic acid makes it ideal for formning complexes with mineral elements, and it has been shown to decrease the availability of calcium, iron, zinc, copper, magnesium and manganese (39) . Dietary fiber in wheat bran includes cellulose, hemicellulose, pectins and lignin‘. Since the results of a recent study (85) have suggested that ionized carboxyl groups of uronic acids can bind to calcium and reduce its availability, it is possible that uronic acids found in specific components of bran fiber might have been responsible for reduced calcium utilization. ' However, since the (diets containing purified fiber sources did not appear 1Unpublished results, based on the analysis done by American Association of Cereal Chemists certified food grade wheat bran (December 1976) . 40 to alter calcium utilization, other factors in bran such as phytates appear to be a more likely cause for the reduced calcium utilization in these animals . When different dietary fiber sources including peanut hulls, corn pericarp and isolated cellulose were given to rats for 18 days, all were shown to increase absorption of radioactive calcium (83) . Contrary to these results increased fecal excretion, negative balance and reduction in serum concentration of calcium have recently bean reported in three human subjects taking 10 grams of cellulose daily for a period of 20 days (81) , suggesting the inhibitory effect of cellulose on calcium availability in man. An inhibitory effect of whole wheat bread on iron absorption has been reported in several studies in human subjects (SS-59) and is also believed to be due to the phytate content of the wheat bran. Reduced serum iron levels in patiants taking 10 to 20 g of bran per day have been reported (62) . The occurrance of iron-deficiency anamia in Iranian villagers cansuming high amounts of unleavened whole mneal bread as the main dietary staple was attributed to the snbstantial amounts of phytate in the bread (59) . Contrary to these findings, Walker 9331. (60) reported no effect of brown bread consnmption on iron retention in humans . In this experimnent bran added extra iron to the basal diet because bran is a rich source of iron. The bran diet contained 93 ppm iron which was considerably higher tlan the amnount in the other diets (54 to 68 ppm) . All diets contained more than the requiramant for growth (35 ppm) of rats recommended by the Natiunal Research Council (86) . Despite higher iron in- takes, bran-fed rats showed similar hematocrit values, tissue concentration and body accnnmlation of iron to those on control and purified fiber diets. Percent body retention relative to intake of iron, however, was significantly 41 lower in animals maintained on the bran diet, and they excreted significantly _ greateramunts of iron in feces during the three days of fecal collection. Availability of the extra iron snpplied by bran would influenCe the interpretation of these results. Morris and Ellis (89) reported that the iron found in bran is primarily in the form of monoferric phytate which has a high biological availability in the rat. If efficiency of iron absorption in bran-fed rats was similar to the rats with lower intakes, the result suggests that perhaps some factors in bran may inhibit irun absorption leading to greater fecal losses , decreased retention relative to intake and no increase in tissue concentration. However, even if the extra iron was available, it may have been absorbed less efficiently because the total intake greatly exceeded the requirement. Similar tissue concentrations and body accumulation in all groups would tend to support such an explanation especially since all intakes were in excess of require- ments. If the extra iron (approximately 44 ppm) supplied by bran was not available, bran did not appear to interfere with the utilization of iron in the bran-free portion of diet (49 ppm) since hematocrit values, tissue concentrations and body accunulation were similar to those sean in other rats ingesting diets with similar or higher (54 to 68 ppm) levels of iron. In contrast to wheat bran, purified fiber sources did not influence iron utilization compared to control . Variations in the amount of intake and fecal excretion of iron and the other trace minerals during the three days of fecal collection resulted in wide ranges of positive and negative apparent absorption within each dietary treatment which decreases the reliability of this method for estimation of percent apparent absorption. Low food intakes and/ or high fecal excre- tion of mnineral in some animals resulted in negative values for apparent absorption of that mineral which does not seem to be reasonable because of 42 the similarity in tissue concentration and body accumulation of mineral in these animals to those having positive apparent absorption. In order to have more hamogenous values and mninimi ze the individual variations in the mineral uptake and excretion, feces should be collected for longer periods of time. Another problem is caprophagy in rats which should be prevented or cunsidered as a source of error. In comparison to a fiber-free diet, diets containing purified fiber did not alter zinc utilization. Tissue concentration, body accumnlation, and percant retention relative to intakes of zinc were similar in all these gromps. Bran supplamentation provided 29 ppm zinc in addition to the amount (17 ppm) contained in the bran-free portion of the diet. This higher dietary level of zinc canbined with greater food consnmption resulted in significantly higher intake of zinc in bran-fed animals. Despite the higher intake, carcass analysis showed significantly lower tissue concantration and body accumulation of zinc in bran-fed animals in Group B. Percent body retantion relative to intake of zinc also was significantly reduced in the bran-fed animals. Even if the extra amount of zinc provided by the wheat bran intake was not in an available form, animals ingested adequate amount of zinc from the bran-free portiun of the diet which was approximately the amnount of zinc ingested by other animals . Therefore, wheat bran appeared to interfere with zinc availability. Reduced zinc availability has bean suggested to be due to the phytate contant of wheat bran (63) . Phytate has bean shown to interfere with mmncosal uptake, binding and absorption of 6"'Zn in rats (71) . A mechanism proposed by Oberleas M. (68, 69) involves the formation of an insoluble salt of phytate in the Lpper intes- tine of rats which is augnented by dietary calcium, forming a Zn-Ca- phytate complex. Impaired zinc retantion also has been reported in human subjects maintained on phytate rich diets (46, 47) . Therefore, the presance 43 of phytate. in bran probably is a reason for the reduced zinc utilization in these animals. Carcass analysis revealed a lower tissue concentration and body accumulation of copper in control animals compared to those on fiber- supplemnented diets . Thus , dietary fiber appeared to augment copper availability. However, bran-fed animals showed significantly lower percent body retention relative to intakes of copper compared to those on purified fiber diets but had similar tissue concentrations and body accumulation of copper. There is little evidence that copper absorption from the intestine is regulated by body needs as is the case for iron. Whan the concentra- tion of capper in the diet is low, absorption exceeds what would be expected on the basis of concentration (90). Normally if the amount of capper in the diet is reduced, a decline in tissue concentration follows. Conversely, if the diet is hign in copper, tissue copper levels, especially in the liver, tend to increase (91) . Bran is a rich source of copper and provided 3.8 ppm copper in addition to the 4.6 ppm supplied by the mineral mix. Since fecal excretion of copper was increased in the bran-fed rats, the extra copper provided by bran may not necessarily have bean available (or totally available) for absorption. If the amonmt of copper provided by the mineral mix is considered as an available source and copper in bran as an unavailable source to the animal, bran supplementation would not appear to have interfered with the availability of copper provided by the mineral mix since tissue copper concantration was not decreased in bran-fed rats. If the cepper provided by wheat bran was in an available form, the significantly higher copper intakes in bran-fed animals would be expected 44 to result in increased tissue copper levels. Because they were similar to those found in rats on purified fiber diets with considerably lower copper intakes, bran supplementation may have interfered with copper availability. Since Davies and Nightingle (71) reported that rats on diets with 10 g added phytate per kg diet showed reduced copper availability, phytate likewise may have been an inhibitory factor on copper utilization in the present experiment . However, the amount of phytate provided by the bran diet was less than the amount fed in the previous study (71) . It is conceivable that components of bran other than phytate also may have been involved in reducing utilization of copper and other minerals. Claims that adaptation to phytate develops in human subjects after a few weeks and balances of calcium and magnesium return to normal if phytate consnmption is prolonged (60) were shown by Reinhold _e_t_§_1_. (46) to be inapplicable to the high intakes brought about by consunption of large amounts of wholemeal bread. When body concentration, accumnlation and retention of minerals in Group A were compared with those in Group B, there was no sign of adaptation to the reduced mineral availability caused by bran and all differences were more significant after a longer period of time (4 weeks compared to 8 weeks). Increased body concentration and accumulation of copper in animals fed purified fiber were statistically significant only after 8 weeks of feeding. Purified fibers, however, did not alter calcium, zinc and iron availability even after a longer period of treatment. Laxative properties of wheat bran have bean known for many years (26, 30, 31). Intakes of 20 g bran per day decreased the transit time in human subjects (29) . In the present study, wheat bran and agar supplemen- tation appeared to cause a more rapid rate of passage of intestinal cantent than the other fibers tested. The percentage of PEG accreted in 32 hours 45 was less in'rats fed guar gmn and MIC—P than in those fed agar and wheat bran but greater than in control animals. Rate of intestinal transit may nnot be related to fecal bulk or water holding capacity alone, but rather to the combination of the two factors. Based on fecal wet and dry weights, the bran diet produced more intestinal residue than the agar diet but the percentage of water content was higher with the agar diet. Both diets, however, produced a similar rate of passage of intestinal contents which was faster than that seen with other diets. An explanation for these results may involve the effect of the specific fiber components in these two diets. Agar is mainly hemicellulose (92) and bran is composed of hemicellulose, cellulose and small amounts of lignin and pectins‘. The presance of hemicellulose in these two diets may have contributed to the faster rate of passage of intestinal contents. Reduced mineral utilization in bran-fed animals did nnot appear to be due to high fecal water holding capacity or fast intestinal transit because animals fed agar had a higher fecal water holding capacity and similar rate of intestinal transit but did not show any impairment in the utiliza- tion of minerals . 1See footnote“) on page 39. WCIUSIGNI Annimals fed diets with added dietary fiber increased their food intakes to provide sufficient energy to maintain weight gains similar to control. Fiber supplementation increased fecal bulk and rate of passage of intes- tinal contents. Fecal water holding capacity likewise was increased by all fiber sources except LIE-P. Bran-fed animals had the highest fecal weight; those fed agar also had high fecal weights and showed the highest fecal water holding capacity. Rate of passage of intestinal contants was faster in rats fed bran and agar than those fed ICC-P and guar gun. Annimals on the fiber-free diet had the lowest fecal weights and the slowest rate of intestinal transit. Dietary intakes of iron, zinc and copper were significantly greater in bran-fed rats because wheat bran is a rich source of these minerals. Despite the higher intakes of calcium and zinc compared to the other animals, tissue concantration and body accumlation of these minerals were signifi- cantly reduced. Bran-fed rats also showed lower concentrations of serum calcium and higher fecal excretion of calcium and zinc. It can be concluded that bran interferes with calcium and zinc utilization. Bran supplementation provided hign intakes of iron, but animals fed bran showed similar hematocrit valnes , tissue concentration and body accumlation of iron to the rats fed the other diets containing less iron. These results maybeexplainedunthebasis ofhigh iron intakeswhichexceededthebody needs of the animals and therefore were not efficiantly absorbed. Reduced 46 47 efficiency of iron absorption also may have been caused by factors suCh as phytate in.the bran diet. Despite the higher intakes of copper provided by bran supplementation, tissue conncentration and body accumulation of copper was similar in rats fed bran and other purified fiber diets. If the amount of copper provided by bran is in.an.availab1e form, then bran :may have interfered with copper utilization. ‘When results in Group A were cunpared with those in Group B, there was no sign of adaptation to the effect of bran on reduced.mdneral availability and all Changes were more pronounced after 8 weeks. Supplementation with purified fiber sources did not alter the calcium, iron and zinc utilization; however, purified fiber appeared to increase cepper utilization. REFERHNICES 10. 11. 12. 13. 14. Burkitt, D. P., Walker, A. R. P. and Painter, N. S. (1974) Dietary fiber and disease. J. Am. Med. Assoc. 229, 1068-1074. Burkitt, D. P. 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(1964) Zinc availability in the chick as affected by phytate, calcium and ethylenediaminetetra- acetate. Poult. Sci. 43, 415-419. Savage, J. E., Yohe, J. M., Pickett, E. E. and O'Dell, B. L. (1964) Zinc metabolism in the growing chick. Tissue concentration and effect of phytate on absorption. Poult. Sci. 43, 420-426. Likuski, H. J. A. and Forbes, R. M. (1964) Effect of phytic acid on the availability of zinc in amino acid and casein diets fed to chicks. J. Nutr. 84, 145-148. Cberleas, D., Muhrer, M. E. and O'Dell, B. L. (1962) Effects of phytic acid on zinc availability and parakeratosis in swine. J. Anim. Sci. 21, 57-61. (berleas, D., Muhrer, M. E. and'O'Dell, B. L. (1966) The availability of zinc from food stuffs. In: Zinc Metabolism (Parasad, A.S., ed) pp. 225—237. Charles C. 'Ihamas, Springfield, Ill. (berleas, D., Muhrer, M. E. and O'Dell, B. L. (1966) Dietary metal- complexing agents and zinc availability in the rat. J. Nutr. 90, 56—62. Pearson, W. N., Schwink. T. and Reich, M. (1966) In vitro studies of zinc absorption in the rat. In: Zinc Metabolism, (Parasad, A.S., ed.) pp. 239-249, Charles C. Thomas, Springfield, Ill. Davies, N. T. and Nightingle, R. (1975) The effects of phytate on intestinal absorption and secretion of zinc, and whole-body retention of zinc, copper, iron and manganese in rats. Brit.J. Nutr. 34, 243—258. Davies, N. T. (1973) Intestinal transport of zinc by rat duodenum. J. Physiol. 229, 46-47. Methfessel, A. H. and Spencer, H. (1973) Zinc metabolism in the rat I. Intestinal absorption of zinc. J. Appl. Physiol. 34, 58-62. 74 . 75. 76. 78. 80. 81. 82. 85. 54 Methfessel, A. H. and Spencer, H. (1973) Zinc metabolism in the rat. II. Secretion of zinc into intestine. J. Appl. Physiol. 34, 63-67. Likuski, H. J. A. and Forbes, R. M. (1965) Mineral utilization in the rat. Effects of calciumand phytic acid on the utilization of dietary zinc. J. Nutr. 85, 230-234. Seeling, M. S. (1964) Perspective in nutrition. The requirerent of magnesium by the normal adults. Am. J. Clin. Nutr. 14, 342-390. Reinhold, J. G., Parsa, A., Karimian, N., Hammick, J. W. and Isnail-Beigi, F. (1974) Availability of zinc in leavened and unleavened wholereal breads as measured by solubility and uptake by rat intestine in vitro. J. Nutr. 104, 976—982. Reinhold, J. G., Israil-Beigi, F. and Faradji, B. (1975) Fiber vs. phytate as determinant of the availability of calcium, zinc and iron of breadstuffs. Nutr. Report Intern. 12, 75-85. Becker, W. M.,and Hoekstra, W. G. (1972) The intestinal absorption of zinc. In: Intestinal absorption of metal ions, trace elerents and radionuclides, (Skoryna, S. C. and Waldron—Edwards, D., eds.) pp. 229-256, Pergammon Press, mford, New York. Reinhold, J. G., Faradji, B., Abadi, P. and Ismail-Beigi, F. (1976) Binding of zinc to fiber and other solids of whole meal bread with a preliminary scandnation of the effects of cellulose consumption upon metabolism of zinc, calcium and phosphorous in man. In: Trace elements in human health and disease, (Parasad, A. S., ed.) pp. 163-180, Academic Press, New York. Isnail-Beigi, F., Reinhold. G. J., Faradji, B. and Abadi, P. (1977) Effects of cellulose added to diets of low and high fiber content upon the metabolism of calcium, magnesium, zinc and phosphorous by men. J. Nutr. 107, 510-518. Pak, C. Y. C., Worstman, J., Bennett, J. E.,Delea, C. S. and Bartter, F. C. (1968) Control of hypercalcenia with cellulose phosphate. J. Clin. Ehdocrinol. 28, 1829-1832. Stiles, L. W., Rivers, J. M., Hackler, L. R., and Vancampen, D. (1976) Altered mineral absorption in the rat due to dietary fiber. Fed. Proc. 35, 744, (Abstract). Viola, S., Zimmermann, G. and Mokady, S. (1970) Effect of pectin and algin upon protein utilization, digestibility of nutrients and energy in young rats. Nutr. Report Intern. 1, 367-375. Branch, W. J., Southgate, D. A. T. and James, W. P. T. (1975) Binding of calcium by dietary fiber: its relationship to unsubstituted uronic acids. Proc. Nutr. Soc. 34, 120A. Cannittee on Animal Nutrition, National Research Comcil. (1972) Nutrient Requirerent of Laboratory Animals. 2nd ed. , National Acadew of Science, Washington, DC. 87. 88. 91. 92. 55 Gill, J. L. Design and Analysis of Experiments in the Animal and Medical sciences. Vol. 1, Iowa State University Press, Ames, Iowa (in press). Hansard, S. L. and Plumlee, M. P. (1954) Effects of dietary calcium and phosphorous levels upon the physiological behavior of calcium and phosphorous in the rat. J. Nutr. 54, 17—31. Morris, E. R. and Ellis, R. ( 1976) Isolation of monoferric phytate from wheat bran and its biological value as an iron source to the rat. J. Nutr. 106, 753-760. Van Campen, D. R. (1971) Absorption of copper from the gastrointestinal tract. In: Intestinal absorption of metal ions, trace elements and radionucleotides, (Skoryna, S. and Waldor-Edward, D. , eds.) pp. 211-227, Pergammon Press, Oxford. Dempsey, H., Cartwright, G. E. and Wintrobe, M. M. (1958) Copper metabolism XXV. Relation between serum and liver copper. Proc. Soc. Exp. Biol. Med. 98, 520-523. Williams, R. D. and Olmsted, W. H. (1936) The effect of cellulose, hemicellulose and lignin on the weight of the stool: A contribu- tion to the study of laxation in man. J. Nutr. 11, 433-449. APPHNIDIX STANDARDS FOR MINERAL ANALYSIS General Reccxmendat ions Use Q acid washed, thoroughly rinsed glassware — including pipettes. Use only deionized distilled water. Use previously labeled primary and working standard polyethylene bottles. Acid wash and rinse well. Do not pipette from primary standard bottles, but pour a quantity into an acid washed beaker and pipette from it into 1 liter volure- tric flask. Be sure to check each new bottle of chemical for the % purity. 57 "Artificial Serum Standard" for Na, K, Cu, Zn, Ca, Mg and P Prepare primary standards first and then use proper amounts for making working standards . Calculations for preparation of primary standards. Sodium: J. T‘3 BAKER REAGENT NaCl (99.5%) dried 2 hr. in vacuum oven at 90 C. MW 58.44 NaCl 1 g Na/lOOO ml=1000 ppm .3934 Na. 25 Na (.3934X=2) 1g NaC1 X Na C1 (Std. 1) X1: 2/.3934 5.0839 = 5.1094 g NaCl .995 (Std. 2) x2= 3/.3934 7.6258 = 7.6641 g NaCl "."99"‘5" (Std. 3) x3= 4/.3934 10.1678 = 10.2189 g NaCl .99" "'5 Add directly to volumetric flask before bringing to volume. Potassium: J. T. BAKER REAGENI‘ KCl (99.5%) dried 2 hrs. in vacuum oven at 90°C. MW KCl = 74.55 39.10/74.55 = .5245 MW K = 39.10 .5245); = 1 g x = _.__1__ = 1.9066 1.9066 = 1.9162 g KCl .5245 ' .995 weigh 1.9162 g KCl, dissolve in HCH and dilute to 1 liter in vol. flask = 1000 ppm Copper: Zinc : Calcium : Magnesium: mosphorus : Weigh 1 gm Cu 100% turnings in an acid washed crucible. Add 5 m1 HNC3 heat (if necessary) to dissolve. Dilute to 1 liter in vol. flask = 1000 ppm Cu J. T. BAKER 100% Zn ribbon weight 1 g Zn metal in an acid washed crucible. Add 5 m1 HCl and heat to dissolve. Dilute to 1 liter in vol. flask = 1000 ppm Zn J. T. BAKER dry 2 hrs. in vacuum oven at 85°C. low in alkalies 99.8% Cam)3 MW Call)3 100.09 40.08/100.29 = .4004 MW Ca 40.08 .4004X = 1 g X = 2.4975 = 2.5025 g Cam3 .998 weigh 2.5025 g c4003, place in vol. flask and add 2-300 m1 Ha-I and 10 ml HCl to dissolve and dilute to 1 liter = 1000 ppm Ca Weigh 1 g Mg slavings in an acid washed crucible. Add 5 ml HCl and heat to dissolve. Dilute to 1 liter in a vol. flask =1000 ppmMg J. T. BAKER ammonium phosphate, dibasic crystals dry in vacuum oven at 80°C for 2 hours. (M1102 H901. (98.0%) F.W. 132.068 30.97/132.1 = .2344 p = 30.97 _ .2344x = 1 g x = 4.2662 = 4.3533 g (NH.,)2HPO., weigh 4.3533 g (NHq)2 E10131, : Dissolve in H20 and dilute to 1 liter in vol. flask = 1000 ppm Working standards . Use following volumes of each primary standard to prepare 1 liter of each working standard (weigh NaCl for each standard). SI‘D.l SID.2 3113.3 5.1094 g 7.6641 g 10.2189 g/liter K 1000 ppm KCl 100 ml 200 ml 300 ml/liter Cu 1000 ppm (turnings) 1 ml 1.5 ml 2.0 ml/liter Zn 1000 ppm (metal) 0.5 ml 1.0 ml 1.5 ml/liter Ca 1000 ppm CaCO3 50 m1 100 ml 150 ml/liter Mg 1000 ppm (shavings) 20 ml 40 ml 60 ml/liter P 1000 ppm (NIIQZHPOL, 25 ml 50 ml 75 ml/liter Final Cation Concentrations as Follows: Sl‘D.l SID.2 SI‘D.3 Na 200ng%or2000ppm 300mg%or3000ppm 400mg%or4000ppm K 10mg%or Cu 100 ug%or Zn 50ug%or Ca 5mg%or Mg 2mg%or P 2.5mg%or 100 plan 1.0 ppm .50 ppm 50 ppm 20 ppm 25ppm 20 mg% or 200 ppm 150 ug% or 1.5 ppm 100 ug% or 1.0 ppm 10 mg% or 100 ppm 4 mg% or 40 ppm 5mg%or 50pm 30mg%or300ppm 200 ug%or2.0ppm 150 ug‘%or1.5ppn 15ng%or150ppm 6mg%or 60pm 7.5 mg%or 75 ppm 61 Feed, Feces and Tissue Standards for Na, K, Cu, Zn, Ca, Mg, P and Fe Prepare primary standards first and then use proper amounts for making working standards. Calculations ‘for preparat ion of primary standards . Sodium: J. T. BAKER REAGENT, NaCl (99.5%) dried in vacuum oven at 90°C for 2 hrs. MW 58.44 NaCl 22.99 _ 22.99 Na 58.44 ' '3934 2.5419 .3934X=1g X=—$5—=2.5546gNacl weigh 2.5546 g NaCl, dissolve in H20 and dilute to 1 liter in vol. flask = 1000 ppm Calcium: J. T. BAKER REAGENT, CaCD (99.9%) dried in vacuum oven at 85°C for 2 hrs. (low in alkalies). MW 100.09 Caoo3 40.08 Ca 40.08/100.09 = .4004 .4004 X = 25 g X = waigh 62.5626 g Gama, place in vol. flask and add 2-300 ml H20 and 10 m1 1121 to dissolve and dilute to 1 liter = 25,0(1) ppm Phosphorus: J. T. BAKER REACEIN'I‘, ammonium phosphate (98.0%) dibasic crystals dried in vacuum oven at 80°C for 2 hrs. MW 132.068 (NH;,)2HPO.+ 30.97 P 30.97 132.068 = '2344 .2344 x = 20 g x = 85.3242 = 87.0655 g "".9“"8 (NH..)2HPOL, 62 weigh 87.0655 g (NHm)2HPC.,, dissolve in H20 and dilute to 1 liter in vol. flask = 20,000 pm Iron: J. T. BAKER REAGENT, FeCl (99.5%) dried in vacuun oven at 90°C for 2 hrs. MW 106.38 FeC13 55.85 Fe 55.85 __ 156.35 ‘ 0'59“" 0.525 X = l g X = 1 = 1.9047 = 1.9142 g FeC13 .525 0.995 weigh 1.9142 g FeCl3, dissolve in H20 and dilute to 1 liter in vol. flask = 1000 ppm Primary standards of K, Cu, Zn andMg areprepared in the same amounts and procedure described for artificial serum standards. Working standards . Use following volumes of each primary standard to prepare 1 liter of each working standard.1 ml/liter SID.1 SID.2 S’ID.3 SI‘D.4 SID.5 Na 1000 ppm NaCl 0.05 0.1 0.2 0.3 0.4 K 1000 ppm KCl 1.0 2.0 3.0 4.0 5.0 Cu 1000 ppm (turnings) 0.1 0.25 0.5 1.0 2.0 Zn 1000 ppm (metal) 0.1 0.25 0.5 1.0 2.0 Ca 25,000 ppm Cath 1.0 2.0 3.0 4.0 5.0 Mg 1000 ppm (shavings) 1.0 2.0 4.0 6.0 8.0 P 20,000 pm(1‘Hu)2HPOI+ 1.0 1.25 2.5 3.75 5.0 Fe 1000 ppm FeC13 1.0 2.0 3.0 4.0 6.0 1Very accurate pipette should be used for slall volures, otherwise further dilutions stould be made to be able to take higher volures and preventing errors. 63 Digest the mixture of standards with concentrated nitric and perchloric acid following the same procedure for digestion of samples. 'Ihem dilute to 1 liter with deionized distilled water. Final Cation Concentrations as Follows: ppm SI‘D.1 S'ID.2 SI‘D.3 8113.4 3113.5 Na 0.05 0.1 0.2 0.3 0.4 K 1 2 3 4 5 Cu 0.1 0.25 0.5 l 2 Zn 0.1 0.25 0.5 1 2 Ca 25 50 75 100 125 Mg 1 2 4 6 8 P 10 25 50 75 100 STATISTICAL EVALUATION (Ire-wags7 analysis of variance (87). In Grow A, a one—way analysis of variance was used to determine statistical differences between mean values for fecal wet and dry weights and water holding capacity. In Grow B, statistical analysis for percent excretion of radioactive PEG was done using this test (Table 16). In both Grows A and B, data for animals fed guar gum diet were included in calculating mean square error. TWO-way analysis of variance (8Q. A two-way factorial design analysis of variance was used to determine differences between means for all data in Grows A and B except those mentioned for one-way analysis of variance. Data for animals fed guar gum diet were excluded in calculating mean square error because these animals were started on diet one week later and killed later. Table 17 is an outline of the analysis for Grows A and B in which a total of 48 animals in two different time intervals were compared. Bonferroni t-stat istics (81). 'Ihree analyses were conducted on each set of data: 1) a carparison between anirmls fed control diet and those fed fiber-swplerented diets (MD-P, agar, guar gun and bran combined); 2) a camparisom between animals fed purified fiber diets (mo-p, agar and guar gum carbined) and bran; and 3) a comparison between animals fed control and those fed purified 64 65 fiber diets (Mm-P, agar and guar gum cmbined). However, in Grow A all comparisons were made with data for the guar gum—fed rats excluded (because those animals were started on diet one week later and sacrificed one week later). Contrasts: K = 1 (Control vs. LIE-P, agar, guar gum and bran combined) K = 2 (Bran vs. Mai-P, agar and guar gum cembined) K = 3 (Control vs. MOI-P, agar and guar gum combined) h The kt . contrast among t means, based on r = 6 observations each represented by: qk= Clkyl."l'C2kyz.+ ..... +Ctkyt, t where 2 C = 0. The tB value used in test was calculated by the 1:1 k following equation: — t =q//(£ C2 1' t3 k i=1 ik/ i)MBE The critical value was obtained fram 1- tB, a/2, m, v which depends won the nurber of contrasts selected (111 = 3), as well as won the total probability of Type I error (on) and nurber of degrees of freeden for experimental error (9). A significant difference at P< «was indicated wher F exceeded the critical value. Stanchrd errors were determined by the following formula: SEM‘W? Table 16. (he-way analysis of variancel. Source of variation Degree of ' Sum of squares Mean square freecbm (SS) (ms) ' t Treatmerts t-l SST=[): y2 /r-(y2 /tr)] NBT= SST/t—l i=1 i' - . Experimental error n-t SSE= SSY-SST MSE= SSE/n-t t r ’1th n-l ssY=[z z y2 -(y2 /tr)] i=1 j=l ij ' ° 1Data of animals fed guar gum diet were included in the analysis (in % PEG excretion and fecal wet and dry weights). There were 5 dietary treat- ments with total of 30 rats ,(6 rats per treatment). TWO-way analysis of variance for Grows A and B. 1 Table 17. Source of variation Degree of freedom Diets (A) 3 Time (B) l Diet-Time 3 Interaction (AB) Error (E) 40 Total (Y) 47 Sun of squares Mean square (38) (me) I. $A=(2 y2 )/12-(y"- /48) SSA/3 i=1 i-- -~- 2 333:“: Y2 )/24-(Y2 /43) 333 3:1 ojo coo ssAB=(§ § y2 )/6-(72 /48) sew/3 i=1 j=l ij. --- ‘SSATSSB ssE= ssY- [SS A+SSB+SS AB] SSE/ 40 1+ 2 6 =