and â¡Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, SK, Canada S7N 5A8. ABSTRACT Broilers chickens have limited ...
Effect of Dietary Calcium, 25-Hydroxycholecalciferol, or Bird Strain on Small Intestinal Phytase Activity in Broiler Chickens1 T. J. Applegate,*,2 R. Angel,† and H. L. Classen‡ *Department of Animal Sciences, Purdue University, West Lafayette, Indiana 47907-1151; †Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland 20742-2311; and ‡Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, SK, Canada S7N 5A8. ABSTRACT Broilers chickens have limited ability to utilize phytate phosphorus (PP), and the influence of nutrients on the activity and efficacy of intestinal phytase is unclear. Therefore in a 2 × 2 × 2 factorial experiment, male chicks were fed 0 or 0.21 mg/kg 25-hydroxycholecalciferol (25-OH D3), 4 or 9 g/kg Ca supplied from CaCO3 or Ca malate from 14 to 24 d of age (six pens/treatment, eight birds/pen). Source of Ca had no effect on tibia ash, intestinal phytase activity, or apparent ileal PP hydrolysis. Phytase activity (Vmax) within brush border vesicles prepared from small intestinal mucosa was greater in chicks fed 4 vs. 9 g/kg Ca (P ≤ 0.05). Similarly, birds fed 4 vs. 9 g/kg Ca were able to hydrolyze an additional 24.36%
PP (P < 0.01). Intestinal phytase activity and apparent ileal PP hydrolysis were not affected by 25-OH D3 supplementation, but tibia ash was improved by 2.7% (P < 0.01). A subsequent experiment validated the effect of dietary Ca (4 and 9 g/kg) and elucidated differences between strains (Ross 308 and Hubbard × Peterson) from 8 to 22 d of age (six pens/treatment, eight birds/pen). The strains responded similarly to dietary Ca in terms of intestinal phytase and apparent ileal PP hydrolysis. Intestinal phytase activity was 9% greater in birds fed 4 vs. 9 g/kg Ca (P < 0.05) and apparent ileal PP hydrolysis was 11.9% greater (P < 0.02). In conclusion, a typical dietary Ca concentration (9 g/kg) reduced intestinal phytase activity and apparent ileal PP hydrolysis.
(Key words: calcium, 25-hydroxycholecalciferol, intestine, phytase, phytate) 2003 Poultry Science 82:1140–1148
INTRODUCTION The ability of phosphorus (P) to be hydrolyzed from phytate within the gastrointestinal tract was first documented in work by Plimmer (1913) with rabbits. In studies with rats, Pattwardhan (1937) attributed this hydrolysis to phytase produced from the small intestine. Subsequent researchers also noted that phytate P (PP) hydrolysis occurred in intestines of chickens, pigs, cows, sheep, and humans (Bitar and Reinhold, 1972). Early methods of intestinal phytase determination consisted of incubating crude mucosal extracts with phytic acid. More recent reports have questioned whether the observed hydrolysis was due to an endogenous mucosal enzyme or if hydrolysis was occurring due to the action of intestinal microbes. Indeed, Wise and Gilburt (1982) reported that PP hydrolysis was negligible for germ-free vs. conventionally reared rats. Similarly, Kerr et al. (2000) reported that PP hydrolysis was
2003 Poultry Science Association, Inc. Received for publication November 22, 2002. Accepted for publication March 26, 2003. 1 Purdue University Agricultural Research Program Jounal Article no. 16937. 2 To whom correspondence should be addressed: applegt@ purdue.edu.
greatly reduced for gnotobiotic chicks. Miyazawa et al. (1996), however, provided data that substantial PP hydrolysis occurred in germ-free and conventionally reared rats, thereby suggesting that intestinal microbes play no major role in PP hydrolysis. To this end, Biehl and Baker (1997) noted substantial hydrolysis of PP from chick mucosal homogenates that contained streptomycin and penicillin to minimize microbial phytase activity. Phytin is often considered to be an antinutrient because of its ability to chelate with dietary cations, rendering the chelated cations partially or completely unavailable to the animal (Pallauf and Rimbach, 1996). Additionally, the chelated mineral-phytin complex is much less soluble at pH values in the small intestine and as such, much less accessible to hydrolysis by exogenous (and presumably endogenous) phytase (Maenz et al., 1999). Negative effects of dietary Ca concentration on PP hydrolysis have been known for some time. For example, Nelson and Kirby (1987) reported an increase in dietary PP hydrolysis from 5.6 to 55% when dietary Ca was reduced from 5.2 to 1.2 g/kg in broiler chicks. Abbreviation Key: 25-OH D3 = 25-hydroxycholecalciferol; BBV = brush border vesicles; ICP = inductively coupled plasma-emission spectroscopy; PP = phytate phosphorus; Vmax = maximum velocity of phytate hydrolysis.
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CALCIUM AFFECTS INTESTINAL PHYTASE ACTIVITY
As growth may not be optimal on low Ca diets, strategies need to be found to deliver Ca to the animal through a means that minimizes Ca-phytin complex formation in the small intestine. Two potential mechanisms to reduce the likelihood for Ca to form complexes with phytin could be utilization of a competitive binding compound and utilization of a compound that would facilitate Ca absorption. Within the intestine, the active vitamin D3 metabolite, 1,25 (OH)2 cholecalciferol, has rapid effects on nonreceptormediated Ca absorption as well as through a slower, receptor-mediated effect on Ca absorption through upregulation of Ca-binding proteins (such as Calbindin D28k; deBoland and Norman, 1990). The commercially available vitamin D3 metabolite, 25-OH cholecalciferol (25-OH D3), may also facilitate an increase in Ca uptake and thus reduce the formation of Ca phytin complexes. An ideally functioning “competitive chelator” would have a higher affinity for mineral binding than would phytate, would be nontoxic, and would allow for ultimate absorption and utilization of the mineral by the animal. Calcium citrate, Ca malate, and Ca citrate-malate have improved solubility characteristics at neutral pH values (Heaney et al., 1990) with a much lower proportion of Ca in the ionic form at pH 7.0 as compared with CaCO3 (RothBassell and Clydesdale, 1992). Additionally, the fractional absorption of Ca and improved bone mineralization from these sources is reportedly greater in rats (Kochanowski, 1990; Andon et al., 1993) and humans (Harvey et al., 1990) than Ca from CaCO3. Therefore the objectives of the current study were to determine if dietary Ca concentration, Ca source, and 25OH D3 supplementation influence intestinal phytase activity and PP hydrolysis within the digestive tract of broiler chicks. As an apparent discrepancy in intestinal phytase responses existed between experiments 1 and 2; a third experiment was conducted to determine whether the discrepancy in Ca response could be attributed to bird strain.
MATERIALS AND METHODS Experiment 1 Research in each experiment was approved by the Purdue Animal Care and Use Committee. From hatching to 7 d of age, birds were fed a diet that met or exceeded all nutrient requirements (NRC, 1994). Hubbard × Peterson, male chicks were fed diets with or without 0.21 mg/kg 25-OH D33 with varying dietary Ca concentrations (4 or 9 g/kg) supplied primarily from CaCO3 from 7 to 21 d of age (five pens/diet, three birds/pen) in brooder battery cages. All birds were fed diets calculated to contain 0.20% nonphytate P. Dietary formulation and selected deter-
3
Hy-D, Monsanto Animal Nutrition, Naperville, IL. Celite Corporation, Lompoc, CA. 5 Central Analytical Laboratory, University of Arkansas, Fayetteville, AR. 6 Sigma-Aldrich Co., St. Louis, MO. 7 Biosoft, Ferguson, MO. 4
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mined analyses are presented in Table 1. Diets contained Celite4 as an inert filler and as a digestive marker at a minimum of 10 g/kg diet to allow for determination of apparent PP hydrolysis. Dietary Ca and total P were determined by inductively coupled plasma-emission spectroscopy (ICP).5 Phytate-P content of diets was determined as described below. At 7 d of age, birds were weighed individually and allocated to experimental pens, such that BW differences were minimized. Bird BW gain and feed consumption were determined from 7 to 21 d of age. At 21 d of age, birds were euthanized by cervical dislocation. The duodenum and jejunum were rinsed in ice-cold saline, and the mucosa was scraped and frozen (−80°C) for future isolation of brush-border vesicles (BBV) and subsequent intestinal phytase determinations (Maenz and Classen, 1998). The digesta in the ileum (as defined by the digesta found between Meckles diverticulum and 2 cm proximal to the ileocecal junction) was removed, placed on ice, and subsequently frozen (−20°C). Mucosa and digesta from birds were pooled by pen. Digesta was lyophilized prior to analyses. Intestinal Phytase Determination. As previous measurements of intestinal phytase were rather controversial, Maenz and Classen (1998) measured true initial rates of PP hydrolysis in preparations of purified chick small intestinal brush-border vesicles. Preparation of the brush-border as the target membrane for activity of the enzyme minimizes complications due to nonbrush border phytases or phosphatases contributing to PP hydrolysis. Therefore, in the current experiments, the method of Maenz and Classen (1998) was used for determination of the specific activity of small intestinal phytase. Briefly, vesicles were produced after precipitation with 1 M MgCl2 and stored in liquid N2 prior to analysis. Aliquots of the final BBV were analyzed for protein concentration by complexing with Coomassie blue and reading the change in absorbance at 595 nm (Sigma protein kit No. 610) utilizing bovine serum albumen as a standard. The BBV were then thawed on ice and aliquoted to microtiter plate wells in triplicate (per phytate concentration) at a concentration of 9 mg protein/mL. Eight phytate (Na phytate from corn6) concentrations were used for determination of intestinal phytase (nmol P released/mg protein per min) kinetics, as follows: 0, 0.025, 0.05, 0.075, 0.1, 0.2, 0.4, and 0.6 mM. Inorganic phosphorus release was determined after 10 min by complexing with ammonium molybdate and reading the absorbance of the reduced complex at 650 nm (Sigma inorganic phosphorus kit No. 670). Kinetic models were tested for convergence (P < 0.05) to nontransformed data obtained for PP hydrolysis with varying substrate concentrations by nonlinear regression analysis and Km and Vmax determined using the Fig P7 program. PP Analyses. Phytate form (myoinositol hexaphosphate to myoinositol diphosphate; IP6 to IP2) quantification was determined as described by Rounds and Nielsen (1993) as modified by Newkirk and Classen (1998). Briefly, samples were extracted with 0.5 N HCl by shaking with an orbital shaker at 2,500 RPM for 3 h. Lipid was then removed from the samples with HPLC-grade chloroform. Extracts were
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APPLEGATE ET AL. TABLE 1. Ingredient and nutrient composition of diets fed to broiler chicks varying in Ca concentration and with or without supplemental 25-hydroxycholecalciferol (25-OH D3), experiment 1 Ingredient, g/kg Corn Soybean meal, 48% CP Canola oil Dicalcium phosphate Limestone Sodium chloride Vitamin-mineral premix1 Choline chloride DL-Methionine L-Lysine Celite2 25-OH vitamin D3 premix3 Dietary analyses, g/kg MEn, kcal/kg, calculated CP, calculated CP, determined Ca, calculated Ca, determined Total P, calculated Total P, determined Phytate P, calculated Phytate P, determined
4 g/kg Ca 586.9 362.6 12.5 3.4 6.4 4.1 5.0 1.0 2.4 0.7 15.0 ... 3,070 220.0 237.4 4.0 4.9 4.7 4.6 2.7 3.1
4 g/kg Ca + 25-OH D3
9 g/kg Ca
585.4 362.6 12.5 3.4 6.4 4.1 5.0 1.0 2.4 0.7 15.0 1.55 3,070 220.0 234.6 4.0 4.7 4.7 4.5 2.7 2.9
560.6 367.6 20.7 3.5 19.5 4.1 5.0 1.0 2.4 0.6 15.0 ... 3,070 220.0 231.8 9.0 9.7 4.6 4.5 2.6 3.0
9 g/kg Ca + 25-OH D3 559.1 367.6 20.7 3.5 19.5 4.1 5.0 1.0 2.4 0.6 15.0 1.55 3,070 220.0 224.0 9.0 10.4 4.6 4.8 2.6 3.1
1 Supplied per kilogram of diet: vitamin A, 11,000 IU; vitamin D3, 2,200 IU; vitamin E, 30 IU; menadione, 0.2 mg; thiamine, 1.5 mg; riboflavin, 6.0 mg; niacin, 60 mg; pyridoxine, 4.0 mg; vitamin B12, 0.02 mg; pantothenic acid, 1.0 mg; folic acid, 0.6 mg; biotin, 0.15 mg; iron, 80 mg; zinc, 80 mg; copper, 10 mg; manganese, 80 mg; iodine, 0.8 mg; and selenium, 0.3 mg. 2 Celite Corporation, Lompoc, CA. 3 25-OH vitamin D3 premix contained 137.8 mg 25-OH vitamin D3 per kilogram. Hy-D, Monsanto Animal Nutrition, Naperville, IL.
analyzed on an HPLC equipped with an anion-exchange column (50 × 4.6 mm PRP X-500 column)8 and a 6.3 × 11.2 mm precolumn. Samples were eluted over a 20 min gradient (0 to 0.5 M NaNO3, 0.01 M 1-methylpiperazine, pH 4.0). The elutant was reacted postcolumn with 0.015% FeCl3 (in 0.15% sulfosalicylic acid), and in-line absorbance was determined at 500 nm. Resultant peak areas were then quantified using a standard curve of peak areas obtained from purified phytic acid.7 Apparent PP hydrolysis determinations were based on acid insoluble ash (AIA) and were conducted with ileal and feed samples according to the procedure of Scott and Boldaji (1997). Apparent PP hydrolysis in ileal digesta was then calculated as [100 − ((AIAdiet × PPdigesta/AIAdigesta × PPdiet) × 100)].
Experiment 2 From hatching to 14 d of age, birds were fed a diet that met or exceeded all nutrient requirements (NRC, 1994). In a 2 × 2 × 2 factorial experiment, Ross 308, male chicks, were fed diets containing 0 or 0.21 mg/kg 25-OH D3, with 4 or 9 g/kg Ca being supplied primarily from either CaCO3 or Ca malate.9 Experimental diets were fed to six replicate pens per diet (eight birds/pen) from 14 to 24 d of age and
birds were housed in brooder-battery cages. All birds were fed diets calculated to contain 0.15% non-phytate P without any supplemental inorganic P. Dietary formulation and selected determined analyses are presented in Table 2. At 14 d of age, birds were weighed individually and allocated to experimental pens, such that BW differences were minimized. Bird BW gain and feed consumption were determined from 14 to 24 d of age. At 24 d of age, birds were euthanized by cervical dislocation. Mucosa and ileal digesta were collected and analyzed as described in experiment 1, with the exception of intestinal phytase data was with Prism10 program for determination of Km and Vmax. The left tibia from three birds per pen (selected at random) was removed. Tibias were then dried for a minimum of 24 h at 100°C, and fat was extracted for a minimum of 8 h with diethyl ether. Ash content of tibias was then determined gravimetrically after ashing in a muffle furnace for 8 h at 600°C. Phytate P analyses and determination of apparent ileal phytate-P hydrolysis were conducted as described in experiment 1. Additionally, diet and ileal contents were analyzed for total P and Ca via ICP as described previously. Apparent ileal Ca and P absorption were calculated as described for apparent PP hydrolysis.
Experiment 3 8
Hamilton, Reno, NV. Monarch Nutritional Laboratories, Ogden, UT. 10 GraphPad Software, Inc., San Diego, CA. 9
Experiment 3 was conducted to elucidate differences in response to dietary Ca between experiments 1 and 2; therefore, different bird strains were used as an experimen-
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CALCIUM AFFECTS INTESTINAL PHYTASE ACTIVITY
TABLE 2. Ingredient and nutrient composition of diets fed to broiler chicks varying in Ca concentration and primary Ca source (CaCO3 or Ca malate) with or without supplemental 25-hydroxycholecalciferol (25-OH D3), experiment 2 4 g/kg Ca CaCO3
Ingredient, g/kg Basal mix1,2 Limestone (CaCO3) Ca malate3 Celite4 25-OH vitamin D3 premix5 Dietary analyses, g/kg MEn, kcal/kg, calculated CP, calculated CP, determined Ca, calculated Ca, determined Total P, calculated Total P, determined Phytate P, calculated Phytate P, determined
970.5 5.0 — 24.6 — 3,060 200 203 4.0 3.62 4.17 3.69 2.71 2.64
4 g/kg Ca CaCO3 + 25-OH D3 970.5 5.0 — 23.0 1.55 3,060 200 209 4.0 3.51 4.17 3.58 2.71 2.65
4 g/kg Ca Ca malate 970.5 — 8.2 21.4 — 3,060 200 207 4.0 3.33 4.17 3.66 2.71 2.67
4 g/kg Ca Ca malate + 25-OH D3
9 g/kg Ca CaCO3
970.5 — 8.2 19.8 1.55 3,060 200 208 4.0 3.30 4.17 3.57 2.71 2.52
970.5 18.0 — 11.6 — 3,060 200 206 9.0 7.61 4.17 3.62 2.71 2.66
9 g/kg Ca CaCO3 + 25-OH D3
9 g/kg Ca Ca malate
970.5 18.0 — 10.0 1.55 3,060 200 208 9.0 8.25 4.17 3.69 2.71 2.56
970.5 — 29.7 8.6 — 3,060 200 205 9.0 7.88 4.17 3.73 2.71 2.47
9 g/kg Ca Ca malate + 25-OH D3 970.5 — 29.7 7.0 1.55 3,060 200 213 9.0 7.87 4.17 3.64 2.71 2.47
1 The basal ration contained the following ingredients (g/kg): corn, 645.1; soybean meal (48% crude protein), 328.1, soy oil, 21.6; DL-methionine, 6.1, salt, 4.1, trace mineral premix, 0.8; vitamin premix, 0.5; and choline chloride (60%), 8. 2 Trace mineral premix and vitamin premix supplied per kilogram of diet: vitamin A, 9,350 IU; vitamin D3, 1,650 IU; vitamin E, 48 IU; menadione, 2.11 mg; thiamine, 6.0 mg; riboflavin, 12.6 mg; niacin, 66.6 mg; pyridoxine, 10.0 mg; vitamin B12, 0.02 mg; pantothenic acid, 22.8 mg; folic acid, 2.0 mg; biotin, 0.23 mg; iron, 164 mg; zinc, 154 mg; copper 18.6 mg; manganese, 80 mg; iodine, 2.7 mg; and selenium, 0.25 mg. 3 Monarch Nutritional Laboratories, Ogden, UT. 4 Celite Corporation, Lompoc, CA. 5 25-OH vitamin D3 premix contained 137.8 mg 25-OH vitamin D3 per kilogram. Hy-D, Monsanto Animal Nutrition, Naperville, IL.
tal variable. From hatching to 8 d of age, birds were fed a diet that met or exceeded all nutrient requirements (NRC, 1994). At 8 d of age, birds were weighed individually and allocated to experimental pens, such that BW differences between pens were minimized. Ross 308 or Hubbard × Peterson, male chicks were fed diets formulated to contain 4 or 9 g/kg Ca supplied from CaCO3 from 8 to 22 d of age (six pens/treatment, eight birds/pen) in electrically heated brooder battery cages. All birds were fed diets calculated to contain 0.18% nonphytate P. Dietary formulation and selected determined analyses are presented in Table 3. Bird BW gain and feed consumption were determined from 8 to 22 d of age. At 22 d of age, birds were euthanized with CO2. Mucosal and ileal digesta were collected and analyzed as described for experiment 2.
Statistical Analyses All experimental data were analyzed statistically by twoway ANOVA using the general linear models procedure of SAS software11 as factorial experiments with all statements of significance ≤ 0.05 unless indicated otherwise.
RESULTS Experiment 1 Chick BW gain was not significantly affected by Ca concentration or 25-OH D3 supplementation from 7 to 21 d of age (P > 0.05; average 21 d BW = 769.7 g; data not shown).
11
SAS Institute, Inc., Cary, NC.
Chicks fed 9 g/kg Ca had significantly greater (18.4 nmol P released/mg protein per min) BBV specific activity of intestinal phytase than chicks fed 4 mg/kg Ca (Vmax and Km; P < 0.05; Table 4). Supplementation with 25-OH D3, however, did not affect intestinal phytase activity. Despite having an increased intestinal phytase activity, chicks fed
TABLE 3. Ingredient and nutrient composition of diets fed to broiler chicks varying in Ca concentration, experiment 3 Ingredient, g/kg Corn Soybean meal, 48% CP Soy oil Dicalcium phosphate Limestone Sodium chloride Vitamin-mineral premix1 DL-Methionine L-Lysine Celite2 Dietary analyses, g/kg MEn, kcal/kg, calculated CP, calculated CP, determined Ca, calculated Ca, determined Total P, calculated Total P, determined Phytate P, calculated Phytate P, determined
4 g/kg Ca
9 g/kg Ca
579.1 345.4 34.0 2.9 6.0 4.1 3.5 1.6 0.2 23.2
579.2 345.4 34.0 2.9 19.2 4.1 3.5 1.6 0.2 10.0
3,070 220 232 4.0 6.14 4.4 4.1 2.6 2.54
3,070 220 232 9.0 11.01 4.4 4.7 2.6 2.85
1 Supplied per kilogram of diet: vitamin A, 13,233 IU; vitamin D3, 3,000 IU; vitamin E, 44 IU; menadione, 4.5 mg; thiamine, 2.2 mg; riboflavin, 6.6 mg; niacin, 88 mg; pyridoxine, 3.3 mg; vitamin B12, 0.02 mg; pantothenic acid, 24 mg; folic acid, 1.1 mg; biotin, choline, 670 mg; 0.33 mg; iron, 50 mg; zinc, 125 mg; copper, manganese, 125.1 mg; 7.7 mg; iodine, 2.1 mg; and selenium, 0.3 mg. 2 Celite Corporation, Lompoc, CA.
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APPLEGATE ET AL. TABLE 4. Small intestinal phytase activity1 and apparent ileal phytate P (PP) digestibility of broiler chicks fed diets varying in Ca concentration and with or without supplemental 25-hydroxycholecalciferol (25-OH D3), experiment 1
Ca (g/kg) 4 9
25-OH D3 (µg/kg)
Km (mM)
0 210 0 210
0.092 0.09 0.12 0.15 0.02
SEM Source of variation Ca concentration 25-OH D3 Ca concentration by 25-OH D3
0.039 0.39 0.32
Main effect means 4 9 0 210
0.09 0.14 0.11 0.12
Vmax (nmol P/mg protein/min) 32.1 27.4 41.9 54.3 6.1 Probability 0.009 0.54 0.18 29.75 48.13 37.03 40.85
Apparent ileal PP hydrolysis (% of PP) 23.97 52.50 32.15 57.21 4.17 0.14 0.0001 0.68 38.23 44.68 28.06 54.86
1 Pooled brush-border vesicles were isolated from pooled mucosa samples by pen. Release of P from eight phytate concentrations (0, 0.025, 0.05, 0.075, 0.1, 0.2, 0.4, and 0.6 mM) were used for fitting of nonlinear regression analyses for determination of Michaelis-Menton enzymatic activity. Km and Vmax indicate the best fit to this model. 2 Means represent five pens of three birds per pen (n = 5).
9 mg/kg Ca were not able to hydrolyze as much PP as indicated by apparent ileal PP hydrolysis (P > 0.05; Table 4). In contrast, supplementation with 0.21 mg/kg 25-OH D3 increased apparent ileal PP hydrolysis by 26.8% (P ≤ 0.0001) despite a lack of effect of 25-OH D3 supplementation on intestinal phytase activity.
Experiment 2 Chick BW gain was not affected by 25-OH D3 supplementation (P > 0.05; Table 5). Birds fed 4 g/kg Ca gained 124.8 g more from 14 to 24 d of age as compared with birds fed 9 g/kg Ca (P ≤ 0.0001). Additionally, birds fed CaCO3 as the primary Ca source gained an additional 10.5 g from 14 to 24 d of age as compared with birds fed Ca malate as the primary Ca source. In contrast to the results of experiment 1, lowering dietary Ca from 9 to 4 g/kg also increased the specific activity of intestinal phytase from 42.48 to 51.66 nmol P released/ mg protein per min, respectively (Vmax; P ≤ 0.05; Table 6). Intestinal phytase activity was unaffected by Ca source (CaCO3 vs. Ca malate) or 25-OH D3 supplementation (0.21 mg/kg). The reduced intestinal phytase activity in birds fed 9 g/ kg Ca corresponded with a 27% reduction in the apparent ileal PP hydrolysis in ileal digesta (P ≤ 0.0001; Table 6) and a 24% reduction in the apparent absorption of P down to the distal ileum (P ≤ 0.0001). No effects of Ca source (CaCO3 vs. Ca malate) or 25-OH D3 supplementation (0.21 mg/ kg) were noted on apparent ileal PP hydrolysis or apparent ileal P absorption (P > 0.05). Apparent ileal Ca absorption was unaffected by Ca source, Ca level, or 25-OH D3 supplementation. The reduced apparent PP hydrolysis and apparent P absorption by the time the digesta reached the distal ileum
from birds fed 9 vs. 4 g/kg Ca also resulted in reduction in tibia ash percentage (41.93 and 46.44%, respectively) and tibia ash weight (533.4 and 720.1 mg/bird, respectively; P ≤ 0.0001; Table 5). Notably, supplementation with 0.21 mg/ kg 25-OH D3 increased tibia ash from 42.82 to 45.55% (P ≤ 0.0001) and tibia ash weight from 607.3 to 646.2 mg/bird (P ≤ 0.063). Ca source, however did not significantly affect tibia ash content.
Experiment 3 Body weight gain was greater in birds fed 4 vs. 9 g/kg Ca (538.7 vs. 508.9 g from 7 to 21 d of age, respectively; P > 0.05; Table 7). Despite having greater specific activity of intestinal phytase, the Hubbard × Peterson birds responded similarly to dietary Ca as the Ross 308 birds as evidenced by the nonsignificant strain by Ca interaction (Table 7). As with experiment 2, birds fed 9 g/kg Ca had significantly reduced BBV specific activity of intestinal phytase (Vmax, and Km; P < 0.05) as compared with chicks fed 4 mg/kg Ca. This corresponded with a reduction in the percentage of apparent PP hydrolysis in ileal digesta (P < 0.02; Table 7). No effects of bird strain or interaction of bird strain by Ca level were noted on apparent ileal PP hydrolysis (P > 0.05).
DISCUSSION Phytin is often thought of as both an antinutritional factor as well as a nutrient. It is often considered toxic, or antinutritive (Pallauf and Rimbach, 1996), because of its ability to chelate to cations in the diet, rendering these chelated cations partially or completely unavailable to the animal. Of the 12 replaceable protons in the phytic acid molecule, six are strongly acidic with a pKa in the range
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CALCIUM AFFECTS INTESTINAL PHYTASE ACTIVITY TABLE 5. Chick BW gain from 14 to 24 d of age and tibia ash content at 24 d of age of broiler chicks fed diets varying in Ca concentration and primary Ca source, with or without supplemental 25-hydroxycholecalciferol (25-OH D3), experiment 2
Ca source CaCO3 CaCO3 CaCO3 CaCO3 Ca Malate Ca Malate Ca Malate Ca Malate SEM
Ca (g/kg)
25-OH D3 (µg/kg)
4 4 9 9 4 4 9 9
0 210 0 210 0 210 0 210
BW gain (g) 503.9 503.9 388.5 380.9 490.0 508.1 364.2 373.0 6.8
1
Tibia ash % 45.75 47.06 40.42 42.95 46.35 46.58 38.74 45.60 0.71
mg/bird 2
709.7 731.8 474.9 536.8 714.1 724.7 530.6 591.5 28.8
Probability Source of variation Ca source Ca concentration 25-OH D3 Ca source × Ca concentration Ca source × 25-OH D3 Ca concentration × 25-OH D3 Ca source × concentration by 25-OH D3
0.036 0.0001 0.32 0.25 0.08 0.38 0.93
Main effect means CaCO3 Ca malate 4 9 0 210
444.3 433.8 501.4 376.6 436.6 441.5
0.58 0.0001 0.0001 0.67 0.11 0.0003 0.01 44.04 44.32 46.44 41.93 42.82 45.55
0.19 0.0001 0.063 0.17 0.88 0.28 0.90 613.3 640.2 720.1 533.4 607.3 646.2
Means represent six pens of six birds per pen (n = 6). Means represent six pens of three birds per pen (n = 6).
1 2
of 1.5 to 2.0, two are weakly acidic with a pKa of approximately 6.0, and four are very weakly acidic with pKa between 9.0 and 11.0 (Costello et al., 1976). Therefore, at all pH values normally encountered in the digestive tract, phytin will carry a strong negative charge and is capable of binding di- and trivalent cations such as Ca, Co, Cu, Fe, Mg, Mn, Ni, and Zn in very stable complexes (Wise, 1983) and reducing the availability of these minerals to the animal (Pallauf and Rimbach, 1996). In experiments 2 and 3 of the current study, apparent ileal PP hydrolysis was increased by 24 and 12%, respectively when dietary Ca was reduced from 9 to 4 g/kg. In experiment 1 however, birds fed the higher Ca concentration (9 g/kg) had increased intestinal phytase activity yet were not able to hydrolyze additional PP when the digesta had reached the distal ileum. The additional endogenous phytase may have been relatively ineffective due to reduced solubility of Ca phytin complexes with the higher dietary Ca concentration. In contrast, the higher endogenous intestinal phytase in experiments 2 and 3 coincided with substantial increases in apparent ileal PP hydrolysis when birds were fed the lower (4 g/kg) Ca concentration. Correlation of intestinal phytase activity (Vmax) with apparent PP hydrolysis was not significant in experiments 1 or 3 but was in experiment 2 (r = 0.31; P ≤ 0.03). Whether the increased hydrolysis in experiments 2 and 3 was directly attributable to the additional intestinal phytase activity, increased solubility of the phytin complex due to reduced Ca phytin complex formation, or both cannot be directly ascertained.
The negative effect of Ca on PP hydrolysis has been noted in previous reports. For example, studies with rats have shown that both age and dietary factors, such as high levels of Ca and P, influence PP hydrolysis in the intestinal tract. Nelson and Kirby (1979) found that weanling rats were capable of digesting 71% of the PP in a corn-soybean meal based diet that contained 0.3% Ca, whereas mature rats utilized only 39% of the PP in the same diet. For both young and mature rats, Ca added to the diet decreased PP hydrolysis. The negative effect of Ca on PP release was confirmed in another study in which rats were shown to utilize only 25% of the PP in a high Ca (1.3%) commercial diet but were capable of hydrolyzing and absorbing 50% of the PP in a low Ca (0.7%) diet (Wise and Gilburt, 1982). Similarly in a study with 3-wk-old broilers, Nelson and Kirby (1987) reported an increase in PP hydrolysis from 5.6 to 55% when chicks were fed a low Ca diet (0.12%) vs. a moderate Ca diet (0.52%). The low dietary Ca levels specified by Nelson and Kirby (1987), however, had an adverse effect on performance and bone mineralization. The reduction of PP hydrolysis with increasing dietary Ca concentrations in experiments 2 and 3 may partially be explained due to formations of insoluble Ca phytin complexes. The increase in pH of the gastrointestinal tract contents as they move distally causes the PP molecule to be ionized and, thus, more readily forms complexes with divalent metal cations such as Zn, Cu, Ca, Mg, and Fe (Wise, 1983). This complexing at higher pH results in decreased solubility of the phytin molecule and increased complex size with increasing concentrations of dietary cat-
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APPLEGATE ET AL. TABLE 6. Intestinal phytase kinetics, apparent ileal phytate P (PP) hydrolysis, and apparent P and Ca absorption at 24 d of age of broiler chicks fed diets varying in Ca concentration and primary Ca source, with or without supplemental 25-hydroxycholecalciferol (25-OH D3), experiment 21
Ca source CaCO3 CaCO3 CaCO3 CaCO3 Ca Malate Ca Malate Ca Malate Ca Malate SEM
Ca (g/kg)
25-OH D3 (µg/kg)
4 4 9 9 4 4 9 9
0 210 0 210 0 210 0 210
Vmax (nmol P released/mg protein/min)
Km (mmol phytate)
47.022 49.61 42.53 42.15 53.61 56.40 41.54 43.96 5.36
0.141 0.169 0.119 0.131 0.146 0.165 0.119 0.124 0.019
Apparent ileal PP hydrolysis (% of total PP) 72.16 75.07 43.49 56.48 71.33 77.77 49.72 49.19 4.98
Apparent ileal P absorption (% of total P) 71.76 71.33 45.14 51.97 73.50 71.78 48.26 46.42 1.93
Apparent ileal Ca absorption (% of total Ca) 68.37 65.20 67.06 67.17 64.90 70.51 65.04 68.79 2.86
Probability Source of variation Ca source Ca concentration 25-OH D3 Ca source × Ca concentration Ca source × 25-OH D3 Ca concentration × 25-OH D3 Ca source × concentration × 25-OH D3 Main effect means CaCO3 Ca malate 4 9 0 210
0.36 0.02 0.63 0.41 0.84 0.83 0.86
0.89 0.02 0.24 0.88 0.75 0.57 0.98
45.32 49.09 51.66 42.48 46.17 48.21
0.140 0.139 0.155 0.123 0.131 0.148
0.95 0.0001 0.13 0.84 0.48 0.83 0.23 61.80 62.00 74.08 49.72 59.18 64.63
0.96 0.0001 0.60 0.40 0.076 0.20 0.18 60.05 59.99 72.09 47.94 59.66 60.38
0.86 0.90 0.44 0.78 0.13 0.86 0.53 66.95 67.31 67.25 67.01 66.34 67.92
1 Pooled brush-border vesicles were isolated from pooled mucosa samples by pen. Release of phosphorus from eight phytate concentrations (0, 0.025, 0.05, 0.075, 0.1, 0.2, 0.4, and 0.6 mM) were used for fitting of nonlinear regression analyses for determination of Michaelis-Menton enzymatic activity. Km and Vmax indicate the best fit to this model. 2 Means represent six pens of six birds per pen (n = 6).
ions, such as Ca (Shafey et al., 1991). The degree to which PP is utilized by an animal will depend, to a large extent, upon its hydrolysis in the gastrointestinal tract. Hydrolysis of PP by phytases occurs when phytin is in solution (O’Dell and deBoland, 1976). Phytin chelates with Ca and other cations and forms insoluble complexes (Wise, 1983) at the higher pH found in the duodenum (pH 5.5), jejunum (pH 6.06), and ileum (pH 6.62) of a chicken (Shafey et al., 1991). Calcium citrate, Ca malate, and Ca citrate-malate have been investigated extensively as Ca supplements. As such, these Ca sources have improved solubility characteristics at neutral pH values in contrast with CaCO3 (Heaney et al., 1990). Additionally, the fractional absorption of Ca and improved bone mineralization from these sources has been notably greater in rats (Kochanowski, 1990; Andon et al., 1993) and humans (Harvey et al., 1990). As such, Lihono et al. (1997) hypothesized that Ca when supplied by Ca citrate-malate may be more resistant to donation of Ca for formation of Ca phytate complexes than Ca from CaCO3. This hypothesis is supported by the fact that Ca citrate and Ca citrate-malate have much lower proportions of Ca in the ionic form at pH 7.0 as compared with CaCO3 (RothBassell and Clydesdale, 1992). In experiment 2 of the current study, Ca source (Ca malate or CaCO3) had no significant effects on any of the parameters measured (apparent ileal PP hydrolysis, P retention, Ca retention, or tibia ash content) in contrast to the reports of Harvey et al. (1990)
and Lihono et al. (1997). Therefore, Ca from either source appears to be equally available for chelation to the phytin molecule in the digestive tract of the chick. Further, Ca from both sources reduced PP hydrolysis (presumably from intestinal phytase or intestinal microorganisms) by 24.36% when Ca content of the diet was increased from 4 to 9 g/kg diet. In experiments 1 and 2, 25-OH D3 supplementation did not significantly affect intestinal phytase activity. Edwards (1993) hypothesized that the 20% improvement in PP hydrolysis when 1,25 (OH)2 vitamin D3 was fed to broiler chicks occurred due either an increase in mucosal phosphate transport or an increase in intestinal phytase activity. Biehl and Baker (1997), however, noted that supplementation with 20 µg/kg of 1α-OH vitamin D3 did not change intestinal phytase activity by the bird nor did addition of 1α-OH vitamin D3 to the mucosal homogenate. Interestingly, 25-OH D3 supplementation improved apparent PP hydrolysis by 27% in experiment 1 and increased bone mineralization in experiment 2 but did not significantly affect apparent ileal PP hydrolysis in experiment 2. Hypothetically, the improvement in PP utilization when 25-OH D3 was supplemented in experiment 1 could have occurred through at least two mechanisms. First, P utilization may have been greater by indirectly improving the rapid phase (transcaltachia) and slower phase (mediated through Ca-binding proteins) Ca uptake from the small
1147
CALCIUM AFFECTS INTESTINAL PHYTASE ACTIVITY 1
TABLE 7. Chick body weight gain from 7 to 21 d of age, intestinal phytase kinetics, and apparent ileal phytate P (PP) hydrolysis at 22 d of age of two strains of broiler chicks fed diets varying in Ca concentration, experiment 3
Strain Ross × Ross Hubbard × Peterson
Calcium (g/kg) 4 9 4 9
SEM Source of variation Strain Calcium level Strain × calcium level
BW gain (g)
Vmax (nmol P released/ mg/protein/min)
539.12 469.9 538.4 520.8 8.6
0.19 0.002 0.17
Main effect means Ross × Ross Hubbard × Peterson 4 9
518.0 529.6 538.7 508.9
Km (mmol phytate)
57.35 49.29 60.02 57.35 2.61
0.198 0.176 0.221 0.196 0.008 Probability
0.05 0.05 0.31
0.019 0.01 0.88
53.32 58.68 58.68 53.32
0.187 0.209 0.210 0.186
Apparent ileal PP hydrolysis (% of total PP) 39.61 22.00 30.74 24.14 4.63
0.47 0.018 0.25 27.44 30.80 35.17 23.07
1 Pooled brush-border vesicles were isolated from pooled mucosa samples by pen. Release of P from eight phytate concentrations (0, 0.025, 0.05, 0.075, 0.1, 0.2, 0.4, and 0.6 mM) were used for fitting of nonlinear regression analyses for determination of Michaelis-Menton enzymatic activity. Km and Vmax indicate the best fit to this model. 2 Means represent six pens of eight birds per pen (n = 6).
intestine (as has been noted for 1,25 (OH)2 cholecalciferol; deBoland and Norman, 1990). As mentioned previously, Ca at pH values in the small intestine can chelate to the phytin molecule and dramatically reduce solubility of phytin molecules and subsequently, hydrolysis by phytase (Maenz et al., 1999). By removing a portion of Ca from the digesta, the phytin molecule is more soluble and accessible to the hydrolytic actions of endogenous (intestinal) or exogenous phytases. As apparent Ca absorption was not measured in experiment 1, we are uncertain as to whether this was a plausible explanation. Apparent Ca absorption, however, was not significantly improved with 25-OH D3 supplementation in experiment 2, but tibia ash content (weight and percentage) was increased with 25-OH D3 supplementation. A second plausible mechanism is that the catalytic actions of some phytases are inhibited by high concentrations of Pi (Wodzinski and Ullah, 1996). As translocation of Pi into the blood from intestinal mucosa is dependant upon vitamin D3 and vitamin D3 metabolites (Wasserman and Taylor, 1973), 25-OH D3 may be assisting the hydrolytic action of phytase by reducing the inhibitory effect of Pi. This may or may not be the case in experiment 1 as apparent P absorption was not measured. However, in experiment 2 in which apparent P absorption was measured, supplementation with 25-OH D3 did not improve apparent ileal absorption of P. The inhibitory effect of Pi on the activity of some phytases has been well documented (Wodzinski and Ullah, 1996). Interestingly, the effect of dietary Ca on PP hydrolysis was more pronounced at lower dietary P concentrations (3.6 g/kg; experiment 2) than at higher dietary P concentrations (4.6 and 4.4 g/kg in experiments 1 and 3, respectively). Whether Pi concentrations within the digesta directly inhibit endogenous phytases (of intestinal or microbial ori-
gin) cannot be directly ascertained through these experiments. Contrary to Motzak (1963), higher concentrations of dietary Ca increased intestinal phytase activity in experiment 1. Reduced activity in experiments 2 and 3 with higher concentrations of dietary Ca were, however, in agreement with Motzak (1963). Experiments 2 and 3 are similar to results reported by McCuaig et al. (1972) who reported that high levels of dietary Mg and Ca reduced both intestinal phytase and alkaline phosphatase activities in the chick. It should be noted, however, that measurement of intestinal phytase in the current studies and that by Motzak (1963) and McCuaig et al. (1972) differ considerably. For example the assay used in the current experiments utilized isolated brush border vesicles isolated from mucosal scrapings, which were frozen upon collection, as opposed to crude mucosal homogenates, which were stored at 5°C for up to 2 wk prior to analyses. Other differences include substrate concentrations and pH conditions at which the assays were conducted. In summary, small intestinal phytase activity of chicks was reduced at “normal” (9 g/kg) dietary Ca concentrations as compared with low dietary Ca concentrations (4 g/ kg) in two of three experiments. This additional intestinal phytase activity coincided with substantial increases in apparent PP hydrolysis within the small intestine. Although 25-OH D3 supplementation increased apparent PP hydrolysis in experiment 1 and bone mineralization in experiment 2, it did not affect intestinal phytase activity in either experiment. Whereas the more soluble Ca source, Ca malate, had been presumed to decrease formation of Ca-phytin complexes at small intestinal pH values, PP hydrolysis was unaffected by Ca source fed in experiment 2. Finally, differences between bird responses to dietary Ca
1148
APPLEGATE ET AL.
concentration in experiments 1 and 2 could not be readily explained by bird strain as evidenced by similar responses to dietary Ca concentration in experiment 3 among strains.
ACKNOWLEDGMENTS The authors thank D. Maenz, R. Newkirk, and C. EngeleSchaan for their assistance with these experiments.
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