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Phytate Content of Excreta and Phytate Retention in the Gastrointestinal Tract of Young Chickens1
Phytate Content of Excreta and Phytate Retention in the Gastrointestinal Tract of Young Chickens1 SOMCHIT SOONCHARERNYING2 and HARDY M. EDWARDS, JR.3 Department of Poultry Science, The University of Georgia, Athens, Georgia 30602
1993 Poultry Science 72:1906-1916
INTRODUCTION The ability of young chicks to use phytate phosphorus is controversial. Lowe et al. (1939) observed a markedly lower bone ash in White Leghorn chicks 5 wk of age receiving diets supplemented with phytate phosphorus compared with chicks fed diets containing inorganic phosphate,
Received for publication February 5, 1993. Accepted for publication June 17, 1993. Supported by state and Hatch funds allocated to the Agricultural Experiment Stations of The University of Georgia. 2 Present address: Conti Chia Tai International Holdings Ltd., Nam Tau, Shenzhen City, China 518052. 3 To whom correspondence should be addressed.
regardless of vitamin D supplementation. Heuser et al. (1943) reported subnormal growth and bone calcification in chicks when phosphorus was supplied mainly as phytate phosphorus in plant feed ingredients and the diet contained adequate levels of vitamin D. McGinnis et al. (1944) showed that phytate phosphorus in some cereals and legumes, including corn, oats, wheat, alfalfa, and soybeans, was not as available as inorganic phosphorus for bone development in young chicks. Other researchers also reported that phytate phosphorus was a poor source of phosphorus for chicks (Gillis et al, 1949, 1957; Ashton et al, 1960; Waldroup et al, 1964; Nelson, 1967). However, the availability of phytate phosphorus was relatively high when chick rations contained either sun-
1906
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ABSTRACT A HPLC procedure was used to determine inositol phosphate in chicken droppings and intestinal content. When compared with the classical ferric chloride method of determining total inositol phosphate in excreta, the HPLC method gave higher values, but the linear relationship between values determined by the two methods was significant (r = .82, P < .014). Holding fecal samples at room temperature (22 C) from 0 to 32 h before analysis for inositol phosphates had no effect on the concentration of inositol hexaphosphate, and, although there were significant effects on inositol pentaphosphate, the results were difficult to interpret. The results indicated very little hydrolysis of inositol phosphate in the excreta of these chickens as they lay on a dropping tray. Excreta from 3-wk-old birds contained significantly less inositol pentaphosphate than excreta from 2-wk-old birds. The excreta from birds receiving a diet containing .27% nonphytate phosphorus contained less inositol pentaphosphate than those receiving a diet containing .42% nonphytate phosphorus. The chromic oxide indicator method seemed to be feasible for determining total phytate disappearance (retention). It was not satisfactory for determining retention in various sections of the gastrointestinal tract, because there seemed to be dilution of phytate and disappearance of chromic oxide in the crop and gizzard in the chicken. There was a marked increase in the concentration of inositol hexaphosphate and to a lesser extent inositol pentaphosphate in the small intestine and cloaca as the other nutrients in the diet were digested. (Key words: chick, age, phytate, inositol hexaphosphate, high-pressure liquid chromatography)
PHYTATE UTILIZATION IN CHICKENS
^Seaboard Farms, Athens, GA 30601.
Subsequently, Sandberg and Ahderinne (1986) developed another modified HPLC method for determination of inositol polyphosphates in foods and in human intestinal contents. These HPLC methods definitely provide a technique capable of determining inositol phosphates as intact molecules. Thus, the utilization of phosphorus in inositol phosphates by poultry can be determined precisely by using the HPLC procedure rather than using the former conventional methods. The purpose of this study was to develop an HPLC procedure to determine phytate content in the diet, intestinal content, and excreta to study the utilization of phytate phosphorus in chickens. Excreta samples were analyzed by the ferric chloride method of Common (1940) and the HPLC method for comparison of the results of the two methods. A study was conducted to determine whether the amount of time the excreta remained in the dropping tray, after defecation by the chicken, had any effect on the amount of phytate found by the HPLC analysis. In addition, a study was conducted to see whether chromic oxide could be used as an indicator to determine phytate phosphorus disappearance from various sections of the gastrointestinal tract of chickens. Both dietary phosphorus level and age of chicken influence phytate utilization. Therefore, the study on disappearance of phytate from excreta on the dropping tray, the study to determine the feasibility of using chromic oxide, and the study of where in the gastrointestinal tract phytate disappears were conducted with diets containing two phosphorus levels and at 1 and 2 wk of age at one of the phosphorus levels. MATERIALS AND METHODS Animals and Treatments Newly hatched Peterson x Arbor Acres cockerels obtained from a commercial hatchery4 were used to evaluate the effects of age and dietary level of phosphorus on phytate content of excreta and phytate retention in the gastrointestinal tract using the chromic oxide method. Group 1 was 120
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cured alfalfa meal (Singsen and Mitchell, 1944) or wheat flour (Sieburth et al, 1952). This may be due to the relatively high content of phytase enzyme in these two ingredients. In most of these studies, phytate or phytic acid utilization has been evaluated by measuring some criterion that responds to dietary phosphorus such as weight gain or bone ash with diets low in phosphorus, for which a response will be obtained from the phosphorus resulting from phytate hydrolysis. A few studies have been reported in which phytate phosphorus retention was calculated from the disappearance of phytate in the excreta as determined by phytate phosphorus analysis of the feed and excreta (Nelson, 1967; Edwards, 1983; Edwards and Veltmann, 1983; Ballam et al, 1984; Elliot and Edwards, 1991a,b). Calcium and phosphorus content of the diet (Edwards and Veltmann, 1983) and age of the chicken (unpublished data) have been shown to influence the utilization of phytate phosphorus from corn-soybean meal diets by broilers. The method of Common (1940) for determining phytate phosphorus has been utilized in most of these studies. This method involves acid extraction followed by the precipitation of an iron complex at low pH. This is followed by the subsequent quantification of phosphorus or iron in the precipitate or analysis of residual iron in the supernatant (Oberleas, 1971). These methods have several disadvantages that may decrease the precision and reproducibility of the results (Graf and Dintzis, 1982). They also do not give any information as to whether the phytate in feed or excreta is all inositol hexaphosphate (IP6) or a mixture of myoinositol 1, 2, 3, 4, 5, 6 hexakis dihydrogen phosphate and what the composition of that mixture might be. Thus, Graf and Dintzis (1982) introduced a simple and reliable HPLC procedure for the determination of myo-inositol 1, 2, 3, 4, 5, 6 hexakis dihydrogen phosphate in plant materials.
1907
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SOONCHARERNYING AND EDWARDS, JR.
Effect of Time of Collection of Excreta After Defecation on Phytate Content In this study, the fecal droppings were collected on plastic trays placed on the dropping pans under the wire floors upon which the chicks stood. At 2 wk of age, plastic trays were placed under 120 chickens, and excreta samples were collected for 10 min. At 3 wk of age, plastic trays were placed under 60 birds receiving each dietary treatment (.27 or .42% nonphytate phosphorus), and excreta samples collected for 10 min. Three excreta samples, approximately 1 g each, were randomly sampled at 0,1,2,4,8,16, and 32 h after collection. Each sample was put in a 20-mL glass vial, immediately frozen in dry ice-acetone bath, and taken to dryness using a freeze dryer6 equipped with an extra acid trap cartridge.6 Dried excreta samples were pulverized
TABLE 1. Composition of the experimental diets Ingredients
Diet 2
Diet 1
lrf\ inn ~\ Kg/ *"» &i
59.83 Ground yellow corn Soybean meal (dehulled) 34.18 1.82 Poultry fat (stabilized) Dicalcium phosphate 1.64 (feed grade) 1.39 Limestone .44 Iodized sodium chloride .25 DL-methionine 1 .25 Vitamin mixture 2 .05 Mineral mixture Se concentrate (.02% .05 from sodium selenite) .10 Chromic oxide 3 Calculated analyses ME, kcal/kg 3,000 Protein, % 22 1.00 Calcium, % Phosphorus .69 Total, % .42 Nonphytate, % .20 Sodium, % 1.23 Lysine, % .95 TSAA, %
60.62 34.04 1.54 .84 1.82 .44 .25 .25 .05 .05 .10
3,000 22 1.00 .54 .27 .20 1.23 .95
'Vitamin mixture provided in milligrams per kilogram of diet (except as noted): all-rrans-retinyl acetate, 1.9; cholecalciferol, 27.5 /*g; all-rac-atocopheryl acetate, 11 IU; riboflavin, 4.4; calcium pantothenate, 12; nicotinic acid, 44; choline CI, 220; vitamin B12, 6.6 /tg; vitamin B& 2.2; menadione (as menadione sodium bisulfite), 1.1; thiamin (as thiamin mononitrate), 2.2; and ethoxyquin, 125. 2 Mineral mixture provided in milligrams per kilogram of diet: Mn (Mn02), 30; Zn (ZnO), 25; Fe (FeS04-7H20), 15; Cu (CuO), 2.5 and I [Ca(I03)2], .75. Calculated from composition tables in Nutrient Requirements of Poultry (NRC, 1984). This NRC publication recommends that 30% of the phosphorus present in plants be considered nonphytate.
using a mortar and pestle, and the ground feed samples were analyzed for inositol phosphates using an HPLC procedure modified from that of Sandberg and Ahderinne (1986). Feed and a composite of the dried excreta samples representing each dietary group treatment were analyzed for Cr 2 0 3 (Brisson, 1956). The ratios of Cr 2 0 3 to phytate in the feed and excreta were used to calculate phytate retention (Edwards and Gillis, 1959). Phytate phosphorus retention is actually the disappearance of phytate phosphorus from the feed at the point it is measured in the gastrointestinal tract or 5 Petersime Incubator Co., Gettysburg, OH 45328. excreta. It is calculated by the equation: 6 The Virtis Co. Inc., Gardiner, NY 12525.
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2-wk-old chicks that had been fed a cornsoybean ration (Table 1) containing .42% nonphytate phosphorus from hatching until 2 wk of age. At 2 wk of age, 60 chicks (Group 2) continued to be fed the diet containing .42% nonphytate phosphorus and 60 chicks (Group 3) were fed the diet containing .27% nonphytate phosphorus until they were 3 wk of age. The 120-day-old commercial broilers that were used to constitute the three groups were initially placed in 12 pens in Petersime batteries5 containing 10 birds per pen. The battery unit was located in a room in which the temperature was maintained at 22 C. Fluorescent lights were on 24 h/day in the room and cages, in the absence of sunlight. Feed and water were provided for ad libitum consumption. After obtaining 2-wk data, the chicks were redistributed into 24 pens containing five birds per pen. Animals were housed and cared for in accordance with guidelines in the Guide for the Care and Use of Laboratory Animals (NRC, 1985) and in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (Consortium, 1988).
PHYTATE UTILIZATION IN CHICKENS
Percentage of phytate phosphorus retained = 100 - percentage Cr 2 0 3 in feed/ percentage Cr 2 0 3 in excreta x percentage phytate phosphorus in excreta/percentage phytate phosphorus in feed x 100. Phytate Content of Digesta In Four Parts of the Gastrointestinal Tract
Phytate Disappearance: Comparing the Ferric Chloride and the HighPerformance Liquid Chromatography Methods
Eight excreta samples representing 24-h collection from chicks fed the diet shown in Table 1 with .42% nonphytate phosphorus were analyzed for phytate by the ferric chloride method (Common, 1940) and the HPLC method to obtain a comparison between the two methods.
(AG1-X8, 200-300 mesh)? used to clean up the sample was increased from .65 to 2.0 mL to give a cleaner excreta inositol phosphate sample for HPLC. After removing the sample from the resin with 20 mL of 2 mol/ L HC1, a fraction of 8 mL of eluent was freeze-dried using the same freeze dryer as mentioned previously. The dried sample was then reconstituted in .8 mL of the mobile phase and 20 pL injected for HPLC analysis. The mobile phase and column were the same as those used by Sandberg and Ahderinne (1986), but the optimal flow rate was 1.5 mL/min under the conditions of the current experiment. Sodium inositol hexaphosphate8 (IP6) was used as both internal and external standards. A commercial phytic acid solution9 previously shown (Lehrfeld, 1989) to contain inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5), and IP6 was also used as a standard solution. The correction values determined by Sandberg and Ahderinne (1986) for differences in refractive index detector response of IP6, IP5, IP4, and IP3 (respectively, 1.0, 1.1, 1.5, and 2.4) were used in all calculations. Differential refractometer10 was used as a detector in these studies. The linearity of phosphate concentration versus peak area was investigated by 20-ftL injections of solutions containing 5.6, 11.2, 16.8, and 22.4 /*g of IP6. Statistical Analysis
Values in the text are means ± SEM. For the statistical analysis of the effect of excreta collection time on the concentration of IP6 in excreta, an analysis of variance and regression analysis were run separately for each of the three groups. Mean differences were separated by Duncan's new multiple range test (Helwig and Council, 1979). To deterAnalytical Methods mine statistical differences among groups, The method described by Sandberg and the data for different times were considered Ahderinne (1986) was used with a few as variable. An analysis of variance of modifications. For the excreta samples, .2 g differences among groups within hour was were used, whereas .5 g of food or intestinal also done for each of the different times. content were used. The amount of resin For the statistical analysis of the retention of IP6 from different sections of the intestinal tract, an analysis of variance was conducted for each of the three groups. To TBio-Rad Laboratories, Richmond, CA 94804. determine statistical differences among sSigma Chemical Co., St. Louis, MO 63178-9916. 9 Aldrich Chemical Co. Inc., Milwaukee, WI 53233. groups within each section, an analysis of variance was done for each section. iOWaters, Milford, MA 01757.
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For the study of retention from various sections of the gastrointestinal tract, nine chicks provided ad libitum access to feed from each of the groups were killed by cervical dislocation, and the contents of the crop, gizzard, distal small intestine, and cloaca were removed. These contents were collected separately from individual chicks and those for three chicks pooled to give a total of three samples from each region of the digestive tract. These samples were then frozen, dried, and pulverized using the same procedure as mentioned previously. Feed and dried samples of gastrointestinal contents were analyzed for Cr 2 0 3 (Brisson, 1956) and inositol phosphates to determine the retention of inositol phosphates in various sections of gastrointestinal tract.
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SOONCHARERNYING AND EDWARDS, JR.
RESULTS
TABLE 2. Analysis of eight excreta samples for inositol phosphates1 by both the ferric chloride and HPLC methods Inositol phytate in excreta Ferric
HPLC method
sample
method
IP4
IPs
IP6
Total
1 2 3 4 5 6 7 8 Mean SEM
13.52 12.4 7.1 9.3 12.5 9.8 8.4 7.5 10.0 .9
.060 .044 .181 .786 .044 .000 .081 .290 .185 .092
1.81 2.02 .16 2.25 1.85 1.40 1.31 1.77 1.57 .23
15.2 15.5 10.1 14.2 16.7 13.0 10.8 12.0 13.4 .8
17.1 17.6 10.4 17.2 18.6 14.4 12.2 14.1 15.2 1.1
!IP4 = inositol tetraphosphate, IP5 = inositol pentaphosphate, IP6 = inositol hexaphosphate. Each value is a single sample.
2
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of IP6 added as an internal standard, and the commercial phytic acid solution (Lehrfeld, 1989) are shown in Figure 1. The chromatoGeneral graphs of the excreta samples and the The results of analyzing eight excreta commercial phytic acid solution show very samples for phytate by the two methods are good separation of the tetra-, penta-, and shown in Table 2. The total phytate ob- hexaphosphoinositol. The commercial tained by the HPLC method was higher in phytic acid solution was analyzed at a all samples than values obtained with the different time from the other three chroferric chloride method. Between 83 and 97% matographs, and the retention time was of the total phytate in the eight excreta longer for all the inositol phosphates. These samples occurred as IP6. All of the excreta different retention times undoubtedly samples except one (Number 3) contained resulted from column age and age of considerable amounts of IP5. Only one of solvent. The effect of both age of column the samples (Number 4) contained signifi- and age of solvent in inositol phosphate cant amounts of IP4. The eight samples had analyses have been discussed by Lehrfeld a range of IP4 from 0 to .786 mg/g with a (1989). mean value of .185 mg/g. There was Recovery studies were conducted in all significant positive correlation (r = .82, P < of the HPLC analyses conducted on excreta .014) between the content of total phytate in and intestinal content samples in the sethe excreta as determined by the ferric quential collection chick experiment and chloride method and the HPLC method. the study on retention of phytate phosphoThe IP4 concentrations for these excreta rus from various sections of the gastrointessamples and those of excreta and gastroin- tinal tract of chickens. The recovery of testinal tract content in the subsequent phytate phosphorus from the excreta colstudies were so low that they were not lected during a 32-h period was between included in calculations of data from the 74.3 and 107.6% with a mean of 87.8 ± 9.1%. other studies. The recovery from samples from the crop, Chromatographs of the IP6 standard, an gizzard, small intestine, and colon ranged excreta sample (Number 4 in Table 2), the from 87.5 to 109.6% with an average of 95.8 same excreta sample with a known amount ± 7.3%.
PHYTATE UTILIZATION IN CHICKENS
1911
Retention of Phytate from Contents of Various Sections of the Gastrointestinal Tract
There was no significant linear or quadratic time effect on IP6 concentration. There was no significant difference in IP6 concentration in excreta due to age and dietary phosphorus. However, the percentage of IP6 retained was significantly different (P > .001) among Groups 1, 2, and 3 (n = 7) and was 22.8 ± 2.9, 32.5 ± 3.4, and 44.6 ± 1.8%, respectively. There was a significant linear and quadratic effect of time on the concentration of IP5 in the excreta of the 3-wk-old birds receiving the diet containing .27% nonphytate phosphorus. The concentration of IP5 decreased with time (Table 3). The excreta of the 2-wk-old birds receiving the .42% nonphytate phosphorus diet had the highest levels of IP5 (4.8 mg/g), followed by the 3-wk-old birds receiving the .42% nonphytate phosphorus diet (3.1 mg/g) and the 3-wk-old birds receiving the .27% nonphytate phosphorus diet (1.6 mg/g).
The amounts of IP6 and IP5 present in the contents from the various sections of the gastrointestinal tract are shown in Table 4. The feed contained 11.4 and .9 mg/g dry matter of IP6 and IP5, respectively. Thus, the amounts of IP6 and IP5 found in the crop and gizzard indicate a decrease in these compounds on a dry matter basis. However, the amounts per gram of dry matter present in the small intestine represents a concentration of the amounts present in the feed. There were a few cases of significant differences between amounts of IP6 and IP5 in certain segments of the gastrointestinal tract as a result of age of chicken and dietary phosphorus levels, however there is no consistent pattern to these differences. Calculated retention percentage of IP6 from different sections of gastrointestinal tract are shown in Table 5. Significant differences in IP6 retention were observed in various parts of the gastrointestinal tract.
TABLE 3. The effect of time that the excreta remained in the dropping tray before freeze drying on the concentration of inositol hexaphosphate (IPg) and inositol pentaphosphate (IP5) in the excreta Time excreta remained on dropping tray before analysis (h) 0 1 2 4 8 16 32 Mean ± SEM3 Probability Linear Quadratic
Group 1 2-wk-old 1 .42% NPP
IP6 Group 2 3-wk-old .42% NPP
IPs Group 1 Group 2 2-wk-old 3-wk-old .42% NPP .42% NPP
Group 3 3-wk-old .27% NPP Kul6'b>
'"
Group 3 3-wk-old .27% NPP
•"
34.22 2.7 1.9 nd 27.1 28.1 26.8 4.9" 31.1 30.9 2.8b 2.0b 31.7 4.9* 34.7 36.7 3.3b 2.0c 32.9a 25.4b 25.7* 5.2" 2.8b 2.0c 27.7 5.5" 28.3 26.5 3.3b .9« b 27.8 27.3 29.0 4.1" 3.1 1.2' 32.2 4.3a 27.5 29.4 3.5b 1.6' 3.1 ± .4b 1.6 ± .2' 4.8 ± .2a 30.9 ± 1.1 29.0 ± 1.3 28.7 ± .7 Regression analysis for IF'6 or IP5 concentration in excreta .254 .418
.349 .378
.696 .366
.340 .705
.418 .758
<.001 .001
a_c Means in a row and IP with no common superscripts differ significantly (P £ .05) based on Duncan's multiple range test. !NPP = nonphytate phosphorus. 2 Values are means (n = 3). 3n = 21.
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Effect of Sequential Collection of Excreta from Dropping Trays on Phytate Content
Dietary nonphytate phosphorus
.005
8.0*3 5.7Y 6.5* 6.7 + .2
IPe
.003
.46 ± .04
.6* .6* .3y
IPs
Crop2
.167
3.0 3.8 2.1 3.0 ± .6
IP 6
<.001
.5" .3y .0* .27 ± .04
IPs
Gizzard IPs
.075 <.001
(mg/g) 2.3* 26.9* .5* 19.6X 1.5y 23.2*y 23.2 ± 1.8 1.43 ± .11
IP6
Small intestine
*- Means in a column with no common superscripts differ significantly (P < .05) based on Duncan's m lr The IP5 values were actually calculated from the response to the IP6 standard in the HPLC procedure. The diet con 2 Number of samples were crop n = 2, gizzard n = 3, small intestine n = 3, and cloaca n = 2. The n for the mean+SEM, cro n = 6. 3 Values are means (n = 3).
z
(wk) (%) .42 2 .42 3 .27 3 Means + SEM ANOVA (Probabilities) df Treatmeni t 2
Age of chick
TABLE 4. Quantities of inositol hexaphosphate (IPg) and inositol pentaphosphate (IPs) sections of the gastrointestinal tract (GIT)1
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PHYTATE UTILIZATION IN CHICKENS
1913
IP6
IP
^
fr^N
El-I B IP6
IP4
IP3 17% 6% as
1PS
33
IP6 44%
J" *;
FIGURE 1. Reverse-phase HPLC of inositol phosphates. A) 16.8 /ig of Sigma (Sigma Chemical Co., St. Louis, MO 63178-9916) inositol hexaphosphate (IP6); B) Excreta sample showing peaks for IP6 at 13.2 jig, inositol pentaphosphate (IP5) at 2.7 jig, inositol tetraphosphate (IF4) at 1.3 /*g, and inositol triphosphate (IP3), quantity cannot be calculated; C) The same fecal sample as B but with a known amount of IP6 added as an internal standard, the increase in peak IP6 should equal 5.7 ng and was equivalent to 6.0 ng (105% recovery); D) The Aldrich (Aldrich Chemical Co., Inc., Milwaukee, WI 53233) commercial phytic acid solution. Lehrfeld (1989) reported a similar solution from Aldrich contained IPg, 43%; IP5,38%; IP4,17%; and IPj, 3%. This chromatograph was obtained at a later date than the other three and the retention times were greater.
The calculated retention from crop and gizzard was higher than that from small intestine and cloaca in all three treatment groups. There was a significant difference in the retention of IP6 in crop among the three treatment groups. The retention was highest in Group 3 and lowest in Group 1 (36 versus 25%). Negative IP6 retention values were calculated for the small intestine and cloaca content; and significantly more IP6 was retained by chickens in Group
3 that received the low-phosphorus diet for 3 wk. In this experiment, chromic oxide concentration varied from 1.20 mg/g of feed to .75 mg/g in gizzard digesta, with ash content increasing from 6.36% in feed to 9.18% in gizzard (Table 6). The decrease in chromic oxide concentration and the increase in ash content in the upper portion of the alimentary tract thus resulted in high retention. A build-up of chromic oxide at
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IP3 IP4
1914
SOONCHARERNYING AND EDWARDS, JR. TABLE 5. Calculated retention of inositol hexaphosphate (IP6) from different sections of gastrointestinal tract Retention of IP 6
Intestinal tract organ
Group 1 2-wk-old .42% NPPi
Group 2 3-wk-old .42% NPP
Group 3 3-wk-old .27% NPP
Crop 2 Gizzard Small intestine Cloaca Means ± SEM
25 b 3 56 -11 -8 16.8 ± 8.4
33 a b 45 -22 -16 10.4 ± 9.8
36" 72 7 4 32.0 ± 7.6
S ± SEM
ANOVA probabilities
31.3 57.6 -8.7 -6.7
.049 .124 .174 .477
IO/\
ab ' Means in a row with no common superscripts differ significantly (P £ .05) based on Duncan's multiple range test. WPP = nonphytate phosphorus. 2 Number of samples were crop n = 2, gizzard n = 3, small intestine n = 3, and cloaca n = 2. The n for mean ± SEM for the three groups n = 10, crop n = 6, gizzard n = 9, small intestine n = 9, and cloaca n = 6. 3 Values are means (n = 3).
the posterior portion therefore caused a negative retention. DISCUSSION
The analysis of excreta samples for phytates reported in Table 2 would indicate in general that the amounts of inositol phosphates other than IP5 and IP6 are present in very low amounts and are probably of little significance in most studies. The analysis of excreta phytate by the ferric chloride method gives lower values in the excreta, which would translate into greater phytate retention when it is calculated from these values. Although it is clear that retention values calculated from the ferric chloride method would be too high, the high correlation obtained between the two methods for just eight samples would still indicate that values obtained with the ferric chloride methods are usable for comparative purposes, such as between dietary treatments. In the sequential collection study, great care was taken in collecting and immediately freeze drying the samples. Thus, it is apparent that no significant conversion of IP6 to phosphate and inositol from either enzymatic or microbial action took place in the excreta on the collection pan at room temperature during this 32-h period after defecation. Under the conditions of this experiment, especially the 22 C temperature of the chick room, it is doubtful
that any microbial action after 32 h would cause a significant increase in the loss of phytate, as the droppings are drying at a very rapid rate at this temperature. It is probably acceptable in phytate retention studies to have collection periods of up to 32 h with little problems from phytate breakdown in the droppings. However, if large amounts of phytase are added to a diet or if ingredients are used that contain considerable amounts of phytase, the collection time may need to be reevaluated. Although age and dietary treatment had a significant effect on the retention of IP6/ there was no significant difference in the IP6 content of the excreta. However, there was a significant difference in the IP5 content of the excreta; the 2-wk-old birds having the highest concentration (4.8 mg/ g) followed by the 3-wk-old birds receiving the diet containing .42% nonphytate P (3.1 mg/g) and the 3-wk-old birds receiving the diet containing .27% nonphytate P (1.6 mg/g). The retention figures confirm work snowing that old birds retain more phytate phosphorus (Edwards et al, 1988) and that increasing the phosphorus content of the diet decreases the utilization of phytate phosphorus (Edwards and Veltmann, 1983). The significant effect of age of birds and dietary nonphytate phosphorus on the IP5 concentration in excreta in the sequential collection study indicates the possible
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± 2.3 ± 7.7 ± 9.5 ± 10.2
PHYTATE UTILIZATION IN CHICKENS
1915
TABLE 6. Ash and chromic oxide concentration in feed and ingesta Variable
Feed
Ash Chromic oxide
6.36 ± 1.131 .120 ± .005a
Crop (g/100 g) ± .9 6 .104 ± .007*
6-86
Gizzard 9.18
± ^52 .075 ± .001b
x ± SEM 7.47 ± 2.60 1.00 ± .009
ANOVA probabilities .296 .002
ab
' Means in a row with no common superscripts differ significantly (P £ .05) based on Duncan's multiple range
test. a
Mean ± SEM, n = 3. Group means ± SEM, n = 9.
the chromic oxide technique to determine phytate hydrolysis in the gastrointestinal tract of the golden hamster and the laboratory rat with no evidence that the chromic oxide underwent any kind of unusual distribution. However, high digestibilities of phytate were found for both animals when the diet contained natural phytase. Negative retention of phytate using the chromic oxide method has previously been reported from this laboratory (Elliot and Edwards, 1991a,b). When samples from a large amount of excreta give a negative value, the most likely explanation is that the analysis of the feed was incorrect and that the value was low, probably due to poor extraction. Another explanation of the high retention of IP6 in the crop and gizzard is suggested by the very low levels of IP6 found in the content of these sections of the tract as compared with the feed. For instance, the gizzard content contained only 3.0 m g / g of IP6 compared with 11.4 m g / g of IP6 in the feed. The most apparent explanation of this is that the soybean meal (high in IP6) is constantly moving out of the crop and gizzard but corn (low in IP6) is remaining. In the present studies with chickens, apparent retention of IP6 in different sections of the alimentary tracts using the chromic oxide indicator method is not a reliable value. These results indicate that the chromic oxide indicator method for determining retention is not a reliable method for detecting quantitative disappearance of IP6 in different sections of the gastrointestinal tract. Chromic oxide previously has been found to be unsatisfactory for determining the absorption of individual fatty acids in the different
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advantage that the HPLC method may have over the ferric chloride method in evaluating nutritional effects on phytate utilization. The lower concentrations of IP5 found in the older birds receiving the diet containing .27% nonphytate phosphorus are of particular interest in view of the 44.6% calculated retention value by this group as compared with 32.5% for birds of the same age fed the .42% nonphytate phosphorus. Thus, the high percentage of phytate phosphorus retention is related to a decrease in the concentration of the first breakdown product (IP5) in the excreta. This may be an indication that a key to the amount of phytate utilized is the amount initially broken down to IP5. There was lower recovery of phytate phosphorus from excreta (87.8%) than from contents of the gastrointestinal tract (95.8%). The most likely explanation of the lower than 100% recovery in both type samples is probably that the IP6 became bound to some material in the samples. However, the possibility that hydrolysis is taking place cannot be ruled out from the information that is available at this time. The calculated retention of IP6 in different sections along the alimentary tract resulted from the change in concentration of IP6 and chromic oxide as the digesta passed down the tract. The high retention of IP6 in crop and gizzard may have resulted from disappearance of chromic oxide from these sections. Johnson et al. (1964) found that chromic oxide in wethers was passed more rapidly out of the anterior portion and less rapidly out of the posterior portion of the digestive tract. This resulted in an increase in the concentration of the indicator in the latter portion. Williams and Taylor (1985) used
1916
SOONCHARERNYING AND EDWARDS, JR.
regions of the digestive system in pigs (Carlson and Bayley, 1968) and in rats (Carlson and Bayley, 1972). However, the chromic oxide indicator method still appears to be a feasible method for the determination of digestibility. REFERENCES
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Ashton, W. M , C. Evans, and P. C. Williams, 1960. Phosphorus compounds of oats. II. The utilisation of phytate phosphorus by growing chicks. J. Sci. Food Agric. 11:722-727. Ballam, G. C , T. S. Nelson, and L. S. Kirby, 1984. Effect of fiber and phytate source and of calcium and phosphorus level on phytate hydrolysis in the chick. Poultry Sci. 63:333-338. Brisson, G. J., 1956. On the routine determination of chromic oxide in feces. Can. J. Agric. Sci. 36: 210-211. Carlson, W. E., and H. S. Bayley, 1968. Utilization of fat by young pigs, fatty acid composition of ingesta in different regions of the digestive tract and apparent and corrected digestibilities of corn oil, lard and tallow. Can. J. Anim. Sci. 48: 315-322. Carlson, W. E., and H. S. Bayley, 1972. Preparation and use of a glyceryl triether as an indicator of fat absorption. Br. J. Nutr. 28:295-305. Common, R. H., 1940. The phytic acid content of some poultry feeding stuffs. Analyst 65:79-83. Consortium, 1988. Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. Association Headquarters, Champaign, IL. Edwards, H. M., Jr., 1983. Phosphorus. 1. Effect of breed and strain on utilization of suboptimal levels of phosphorus in the ration. Poultry Sci. 62:77-84. Edwards, H. M , Jr., 1988. Effect of dietary calcium, phosphorus, chloride, and zeolite on the development of tibial dyschondroplasia. Poultry Sci. 67:1436-1446. Edwards, H. M., Jr., and M. B. Gillis, 1959. A chromic oxide balance method for determining phosphate availability. Poultry Sci. 38:569-574. Edwards, H. M., Jr., and J. R. Veltmann, Jr., 1983. The role of calcium and phosphorus in the etiology of tibial dyschondroplasia in young chicks. J. Nutr. 113:1568-1575. Elliot, M. A., and H. M. Edwards, Jr., 1991a. Some effects of dietary aluminum and silicon on broiler chickens. Poultry Sci. 70:1390-1402. Elliot, M. A., and H. M. Edwards, Jr., 1991b. Comparison of the effects of synthetic and natural zeolite on laying hen and broiler chicken performance. Poultry Sci. 70:2115-2130.
Gillis, M. B., K. W. Keane, and R. A. Collins, 1957. Comparative metabolism of phytate and inorganic P 3 2 by chicks and poults. J. Nutr. 62:13-26. Gillis, M. B., L. C. Norris, and G. F. Heuser, 1949. The effect of phytin on the phosphorus requirement of the chick. Poultry Sci. 28:283-288. Graf, E., and F. R. Dintzis, 1982. High-performance liquid chromatographic method for the determination of phytate. Anal. Biochem. 119:413-417. Helwig, J. T., and K. A. Council, 1979. SAS® User's Guide, 1979 ed. SAS Institute Inc., Raleigh, NC. Heuser, G. F., L. C. Norris, J. McGinnis, and M. L. Scott, 1943. Further evidence of the need for supplementing soybean meal chick rations with phosphorus. Poultry Sci. 22:269-270. Johnson, D. E., W. E. Dinersson, and D. W. Bolin, 1964. Rate of passage of chromic oxide and composition of digesta along the alimentary tract of wethers. J. Anim. Sci. 23:499-505. Lehrfeld, J., 1989. High-performance liquid chromatography analysis of phytic acid on a pHstable, macroporous polymer column. Cereal Chem. 66(6)510-515. Lowe, J. T., H. Steenbock, and C. H. Krieger, 1939. Cereals and rickets. IX. The availability of phytin-P to the chick. Poultry Sci. 18:40-44. McGinnis, J., L. C. Norris, and G. F. Heuser, 1944. Poor utilization of phosphorus in cereals and legumes by chicks for bone development. Poultry Sci. 23:157-159. National Research Council, 1985. Guide for the Care and Use of Laboratory Animals. National Institutes of Health Publication No. 85-23, rev. National Institutes of Health, Bethesda, MD. Nelson, T. S., 1967. The utilization of phytate phosphorus by poultry—A review. Poultry Sci. 46:862-871. Oberleas, D., 1971. The determination of phytate and inositol phosphates. Methods Biochem. Anal. 20:87-101. Sandberg, A. S., and R. Ahderinne, 1986. HPLC method for determination of inositol tri-, terra-, penta-, and hexaphosphates in foods and intestinal contents. J. Food Sci. 51:547-550. Sieburth, J. F., J. McGinnis, T. Wahl, and B. A. McLaren, 1952. The availability of the phosphorus in unifine flour for the chick. Poultry Sci. 31: 813-818. Singsen, E. P., and H. H. Mitchell, 1944. Soybean meal chick rations need no inorganic phosphorus supplements. Poultry Sci. 23:152-153. Waldroup, P. W., C. B. Ammerman, and R. H. Harms, 1964. The availability of phytic acid phosphorus for chicks. 2. Comparison of phytin phosphorus sources. Poultry Sci. 43:426-432. Williams, P. J., and T. G. Taylor, 1985. A comparative study of phytate hydrolysis in the gastrointestinal tract of the golden hamster (Mesocricetus auratus) and the laboratory rat. Br. J. Nutr. 54: 429-435.
1993 Poultry Science 72:1906-1916
INTRODUCTION The ability of young chicks to use phytate phosphorus is controversial. Lowe et al. (1939) observed a markedly lower bone ash in White Leghorn chicks 5 wk of age receiving diets supplemented with phytate phosphorus compared with chicks fed diets containing inorganic phosphate,
Received for publication February 5, 1993. Accepted for publication June 17, 1993. Supported by state and Hatch funds allocated to the Agricultural Experiment Stations of The University of Georgia. 2 Present address: Conti Chia Tai International Holdings Ltd., Nam Tau, Shenzhen City, China 518052. 3 To whom correspondence should be addressed.
regardless of vitamin D supplementation. Heuser et al. (1943) reported subnormal growth and bone calcification in chicks when phosphorus was supplied mainly as phytate phosphorus in plant feed ingredients and the diet contained adequate levels of vitamin D. McGinnis et al. (1944) showed that phytate phosphorus in some cereals and legumes, including corn, oats, wheat, alfalfa, and soybeans, was not as available as inorganic phosphorus for bone development in young chicks. Other researchers also reported that phytate phosphorus was a poor source of phosphorus for chicks (Gillis et al, 1949, 1957; Ashton et al, 1960; Waldroup et al, 1964; Nelson, 1967). However, the availability of phytate phosphorus was relatively high when chick rations contained either sun-
1906
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ABSTRACT A HPLC procedure was used to determine inositol phosphate in chicken droppings and intestinal content. When compared with the classical ferric chloride method of determining total inositol phosphate in excreta, the HPLC method gave higher values, but the linear relationship between values determined by the two methods was significant (r = .82, P < .014). Holding fecal samples at room temperature (22 C) from 0 to 32 h before analysis for inositol phosphates had no effect on the concentration of inositol hexaphosphate, and, although there were significant effects on inositol pentaphosphate, the results were difficult to interpret. The results indicated very little hydrolysis of inositol phosphate in the excreta of these chickens as they lay on a dropping tray. Excreta from 3-wk-old birds contained significantly less inositol pentaphosphate than excreta from 2-wk-old birds. The excreta from birds receiving a diet containing .27% nonphytate phosphorus contained less inositol pentaphosphate than those receiving a diet containing .42% nonphytate phosphorus. The chromic oxide indicator method seemed to be feasible for determining total phytate disappearance (retention). It was not satisfactory for determining retention in various sections of the gastrointestinal tract, because there seemed to be dilution of phytate and disappearance of chromic oxide in the crop and gizzard in the chicken. There was a marked increase in the concentration of inositol hexaphosphate and to a lesser extent inositol pentaphosphate in the small intestine and cloaca as the other nutrients in the diet were digested. (Key words: chick, age, phytate, inositol hexaphosphate, high-pressure liquid chromatography)
PHYTATE UTILIZATION IN CHICKENS
^Seaboard Farms, Athens, GA 30601.
Subsequently, Sandberg and Ahderinne (1986) developed another modified HPLC method for determination of inositol polyphosphates in foods and in human intestinal contents. These HPLC methods definitely provide a technique capable of determining inositol phosphates as intact molecules. Thus, the utilization of phosphorus in inositol phosphates by poultry can be determined precisely by using the HPLC procedure rather than using the former conventional methods. The purpose of this study was to develop an HPLC procedure to determine phytate content in the diet, intestinal content, and excreta to study the utilization of phytate phosphorus in chickens. Excreta samples were analyzed by the ferric chloride method of Common (1940) and the HPLC method for comparison of the results of the two methods. A study was conducted to determine whether the amount of time the excreta remained in the dropping tray, after defecation by the chicken, had any effect on the amount of phytate found by the HPLC analysis. In addition, a study was conducted to see whether chromic oxide could be used as an indicator to determine phytate phosphorus disappearance from various sections of the gastrointestinal tract of chickens. Both dietary phosphorus level and age of chicken influence phytate utilization. Therefore, the study on disappearance of phytate from excreta on the dropping tray, the study to determine the feasibility of using chromic oxide, and the study of where in the gastrointestinal tract phytate disappears were conducted with diets containing two phosphorus levels and at 1 and 2 wk of age at one of the phosphorus levels. MATERIALS AND METHODS Animals and Treatments Newly hatched Peterson x Arbor Acres cockerels obtained from a commercial hatchery4 were used to evaluate the effects of age and dietary level of phosphorus on phytate content of excreta and phytate retention in the gastrointestinal tract using the chromic oxide method. Group 1 was 120
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cured alfalfa meal (Singsen and Mitchell, 1944) or wheat flour (Sieburth et al, 1952). This may be due to the relatively high content of phytase enzyme in these two ingredients. In most of these studies, phytate or phytic acid utilization has been evaluated by measuring some criterion that responds to dietary phosphorus such as weight gain or bone ash with diets low in phosphorus, for which a response will be obtained from the phosphorus resulting from phytate hydrolysis. A few studies have been reported in which phytate phosphorus retention was calculated from the disappearance of phytate in the excreta as determined by phytate phosphorus analysis of the feed and excreta (Nelson, 1967; Edwards, 1983; Edwards and Veltmann, 1983; Ballam et al, 1984; Elliot and Edwards, 1991a,b). Calcium and phosphorus content of the diet (Edwards and Veltmann, 1983) and age of the chicken (unpublished data) have been shown to influence the utilization of phytate phosphorus from corn-soybean meal diets by broilers. The method of Common (1940) for determining phytate phosphorus has been utilized in most of these studies. This method involves acid extraction followed by the precipitation of an iron complex at low pH. This is followed by the subsequent quantification of phosphorus or iron in the precipitate or analysis of residual iron in the supernatant (Oberleas, 1971). These methods have several disadvantages that may decrease the precision and reproducibility of the results (Graf and Dintzis, 1982). They also do not give any information as to whether the phytate in feed or excreta is all inositol hexaphosphate (IP6) or a mixture of myoinositol 1, 2, 3, 4, 5, 6 hexakis dihydrogen phosphate and what the composition of that mixture might be. Thus, Graf and Dintzis (1982) introduced a simple and reliable HPLC procedure for the determination of myo-inositol 1, 2, 3, 4, 5, 6 hexakis dihydrogen phosphate in plant materials.
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SOONCHARERNYING AND EDWARDS, JR.
Effect of Time of Collection of Excreta After Defecation on Phytate Content In this study, the fecal droppings were collected on plastic trays placed on the dropping pans under the wire floors upon which the chicks stood. At 2 wk of age, plastic trays were placed under 120 chickens, and excreta samples were collected for 10 min. At 3 wk of age, plastic trays were placed under 60 birds receiving each dietary treatment (.27 or .42% nonphytate phosphorus), and excreta samples collected for 10 min. Three excreta samples, approximately 1 g each, were randomly sampled at 0,1,2,4,8,16, and 32 h after collection. Each sample was put in a 20-mL glass vial, immediately frozen in dry ice-acetone bath, and taken to dryness using a freeze dryer6 equipped with an extra acid trap cartridge.6 Dried excreta samples were pulverized
TABLE 1. Composition of the experimental diets Ingredients
Diet 2
Diet 1
lrf\ inn ~\ Kg/ *"» &i
59.83 Ground yellow corn Soybean meal (dehulled) 34.18 1.82 Poultry fat (stabilized) Dicalcium phosphate 1.64 (feed grade) 1.39 Limestone .44 Iodized sodium chloride .25 DL-methionine 1 .25 Vitamin mixture 2 .05 Mineral mixture Se concentrate (.02% .05 from sodium selenite) .10 Chromic oxide 3 Calculated analyses ME, kcal/kg 3,000 Protein, % 22 1.00 Calcium, % Phosphorus .69 Total, % .42 Nonphytate, % .20 Sodium, % 1.23 Lysine, % .95 TSAA, %
60.62 34.04 1.54 .84 1.82 .44 .25 .25 .05 .05 .10
3,000 22 1.00 .54 .27 .20 1.23 .95
'Vitamin mixture provided in milligrams per kilogram of diet (except as noted): all-rrans-retinyl acetate, 1.9; cholecalciferol, 27.5 /*g; all-rac-atocopheryl acetate, 11 IU; riboflavin, 4.4; calcium pantothenate, 12; nicotinic acid, 44; choline CI, 220; vitamin B12, 6.6 /tg; vitamin B& 2.2; menadione (as menadione sodium bisulfite), 1.1; thiamin (as thiamin mononitrate), 2.2; and ethoxyquin, 125. 2 Mineral mixture provided in milligrams per kilogram of diet: Mn (Mn02), 30; Zn (ZnO), 25; Fe (FeS04-7H20), 15; Cu (CuO), 2.5 and I [Ca(I03)2], .75. Calculated from composition tables in Nutrient Requirements of Poultry (NRC, 1984). This NRC publication recommends that 30% of the phosphorus present in plants be considered nonphytate.
using a mortar and pestle, and the ground feed samples were analyzed for inositol phosphates using an HPLC procedure modified from that of Sandberg and Ahderinne (1986). Feed and a composite of the dried excreta samples representing each dietary group treatment were analyzed for Cr 2 0 3 (Brisson, 1956). The ratios of Cr 2 0 3 to phytate in the feed and excreta were used to calculate phytate retention (Edwards and Gillis, 1959). Phytate phosphorus retention is actually the disappearance of phytate phosphorus from the feed at the point it is measured in the gastrointestinal tract or 5 Petersime Incubator Co., Gettysburg, OH 45328. excreta. It is calculated by the equation: 6 The Virtis Co. Inc., Gardiner, NY 12525.
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2-wk-old chicks that had been fed a cornsoybean ration (Table 1) containing .42% nonphytate phosphorus from hatching until 2 wk of age. At 2 wk of age, 60 chicks (Group 2) continued to be fed the diet containing .42% nonphytate phosphorus and 60 chicks (Group 3) were fed the diet containing .27% nonphytate phosphorus until they were 3 wk of age. The 120-day-old commercial broilers that were used to constitute the three groups were initially placed in 12 pens in Petersime batteries5 containing 10 birds per pen. The battery unit was located in a room in which the temperature was maintained at 22 C. Fluorescent lights were on 24 h/day in the room and cages, in the absence of sunlight. Feed and water were provided for ad libitum consumption. After obtaining 2-wk data, the chicks were redistributed into 24 pens containing five birds per pen. Animals were housed and cared for in accordance with guidelines in the Guide for the Care and Use of Laboratory Animals (NRC, 1985) and in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (Consortium, 1988).
PHYTATE UTILIZATION IN CHICKENS
Percentage of phytate phosphorus retained = 100 - percentage Cr 2 0 3 in feed/ percentage Cr 2 0 3 in excreta x percentage phytate phosphorus in excreta/percentage phytate phosphorus in feed x 100. Phytate Content of Digesta In Four Parts of the Gastrointestinal Tract
Phytate Disappearance: Comparing the Ferric Chloride and the HighPerformance Liquid Chromatography Methods
Eight excreta samples representing 24-h collection from chicks fed the diet shown in Table 1 with .42% nonphytate phosphorus were analyzed for phytate by the ferric chloride method (Common, 1940) and the HPLC method to obtain a comparison between the two methods.
(AG1-X8, 200-300 mesh)? used to clean up the sample was increased from .65 to 2.0 mL to give a cleaner excreta inositol phosphate sample for HPLC. After removing the sample from the resin with 20 mL of 2 mol/ L HC1, a fraction of 8 mL of eluent was freeze-dried using the same freeze dryer as mentioned previously. The dried sample was then reconstituted in .8 mL of the mobile phase and 20 pL injected for HPLC analysis. The mobile phase and column were the same as those used by Sandberg and Ahderinne (1986), but the optimal flow rate was 1.5 mL/min under the conditions of the current experiment. Sodium inositol hexaphosphate8 (IP6) was used as both internal and external standards. A commercial phytic acid solution9 previously shown (Lehrfeld, 1989) to contain inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5), and IP6 was also used as a standard solution. The correction values determined by Sandberg and Ahderinne (1986) for differences in refractive index detector response of IP6, IP5, IP4, and IP3 (respectively, 1.0, 1.1, 1.5, and 2.4) were used in all calculations. Differential refractometer10 was used as a detector in these studies. The linearity of phosphate concentration versus peak area was investigated by 20-ftL injections of solutions containing 5.6, 11.2, 16.8, and 22.4 /*g of IP6. Statistical Analysis
Values in the text are means ± SEM. For the statistical analysis of the effect of excreta collection time on the concentration of IP6 in excreta, an analysis of variance and regression analysis were run separately for each of the three groups. Mean differences were separated by Duncan's new multiple range test (Helwig and Council, 1979). To deterAnalytical Methods mine statistical differences among groups, The method described by Sandberg and the data for different times were considered Ahderinne (1986) was used with a few as variable. An analysis of variance of modifications. For the excreta samples, .2 g differences among groups within hour was were used, whereas .5 g of food or intestinal also done for each of the different times. content were used. The amount of resin For the statistical analysis of the retention of IP6 from different sections of the intestinal tract, an analysis of variance was conducted for each of the three groups. To TBio-Rad Laboratories, Richmond, CA 94804. determine statistical differences among sSigma Chemical Co., St. Louis, MO 63178-9916. 9 Aldrich Chemical Co. Inc., Milwaukee, WI 53233. groups within each section, an analysis of variance was done for each section. iOWaters, Milford, MA 01757.
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For the study of retention from various sections of the gastrointestinal tract, nine chicks provided ad libitum access to feed from each of the groups were killed by cervical dislocation, and the contents of the crop, gizzard, distal small intestine, and cloaca were removed. These contents were collected separately from individual chicks and those for three chicks pooled to give a total of three samples from each region of the digestive tract. These samples were then frozen, dried, and pulverized using the same procedure as mentioned previously. Feed and dried samples of gastrointestinal contents were analyzed for Cr 2 0 3 (Brisson, 1956) and inositol phosphates to determine the retention of inositol phosphates in various sections of gastrointestinal tract.
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SOONCHARERNYING AND EDWARDS, JR.
RESULTS
TABLE 2. Analysis of eight excreta samples for inositol phosphates1 by both the ferric chloride and HPLC methods Inositol phytate in excreta Ferric
HPLC method
sample
method
IP4
IPs
IP6
Total
1 2 3 4 5 6 7 8 Mean SEM
13.52 12.4 7.1 9.3 12.5 9.8 8.4 7.5 10.0 .9
.060 .044 .181 .786 .044 .000 .081 .290 .185 .092
1.81 2.02 .16 2.25 1.85 1.40 1.31 1.77 1.57 .23
15.2 15.5 10.1 14.2 16.7 13.0 10.8 12.0 13.4 .8
17.1 17.6 10.4 17.2 18.6 14.4 12.2 14.1 15.2 1.1
!IP4 = inositol tetraphosphate, IP5 = inositol pentaphosphate, IP6 = inositol hexaphosphate. Each value is a single sample.
2
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of IP6 added as an internal standard, and the commercial phytic acid solution (Lehrfeld, 1989) are shown in Figure 1. The chromatoGeneral graphs of the excreta samples and the The results of analyzing eight excreta commercial phytic acid solution show very samples for phytate by the two methods are good separation of the tetra-, penta-, and shown in Table 2. The total phytate ob- hexaphosphoinositol. The commercial tained by the HPLC method was higher in phytic acid solution was analyzed at a all samples than values obtained with the different time from the other three chroferric chloride method. Between 83 and 97% matographs, and the retention time was of the total phytate in the eight excreta longer for all the inositol phosphates. These samples occurred as IP6. All of the excreta different retention times undoubtedly samples except one (Number 3) contained resulted from column age and age of considerable amounts of IP5. Only one of solvent. The effect of both age of column the samples (Number 4) contained signifi- and age of solvent in inositol phosphate cant amounts of IP4. The eight samples had analyses have been discussed by Lehrfeld a range of IP4 from 0 to .786 mg/g with a (1989). mean value of .185 mg/g. There was Recovery studies were conducted in all significant positive correlation (r = .82, P < of the HPLC analyses conducted on excreta .014) between the content of total phytate in and intestinal content samples in the sethe excreta as determined by the ferric quential collection chick experiment and chloride method and the HPLC method. the study on retention of phytate phosphoThe IP4 concentrations for these excreta rus from various sections of the gastrointessamples and those of excreta and gastroin- tinal tract of chickens. The recovery of testinal tract content in the subsequent phytate phosphorus from the excreta colstudies were so low that they were not lected during a 32-h period was between included in calculations of data from the 74.3 and 107.6% with a mean of 87.8 ± 9.1%. other studies. The recovery from samples from the crop, Chromatographs of the IP6 standard, an gizzard, small intestine, and colon ranged excreta sample (Number 4 in Table 2), the from 87.5 to 109.6% with an average of 95.8 same excreta sample with a known amount ± 7.3%.
PHYTATE UTILIZATION IN CHICKENS
1911
Retention of Phytate from Contents of Various Sections of the Gastrointestinal Tract
There was no significant linear or quadratic time effect on IP6 concentration. There was no significant difference in IP6 concentration in excreta due to age and dietary phosphorus. However, the percentage of IP6 retained was significantly different (P > .001) among Groups 1, 2, and 3 (n = 7) and was 22.8 ± 2.9, 32.5 ± 3.4, and 44.6 ± 1.8%, respectively. There was a significant linear and quadratic effect of time on the concentration of IP5 in the excreta of the 3-wk-old birds receiving the diet containing .27% nonphytate phosphorus. The concentration of IP5 decreased with time (Table 3). The excreta of the 2-wk-old birds receiving the .42% nonphytate phosphorus diet had the highest levels of IP5 (4.8 mg/g), followed by the 3-wk-old birds receiving the .42% nonphytate phosphorus diet (3.1 mg/g) and the 3-wk-old birds receiving the .27% nonphytate phosphorus diet (1.6 mg/g).
The amounts of IP6 and IP5 present in the contents from the various sections of the gastrointestinal tract are shown in Table 4. The feed contained 11.4 and .9 mg/g dry matter of IP6 and IP5, respectively. Thus, the amounts of IP6 and IP5 found in the crop and gizzard indicate a decrease in these compounds on a dry matter basis. However, the amounts per gram of dry matter present in the small intestine represents a concentration of the amounts present in the feed. There were a few cases of significant differences between amounts of IP6 and IP5 in certain segments of the gastrointestinal tract as a result of age of chicken and dietary phosphorus levels, however there is no consistent pattern to these differences. Calculated retention percentage of IP6 from different sections of gastrointestinal tract are shown in Table 5. Significant differences in IP6 retention were observed in various parts of the gastrointestinal tract.
TABLE 3. The effect of time that the excreta remained in the dropping tray before freeze drying on the concentration of inositol hexaphosphate (IPg) and inositol pentaphosphate (IP5) in the excreta Time excreta remained on dropping tray before analysis (h) 0 1 2 4 8 16 32 Mean ± SEM3 Probability Linear Quadratic
Group 1 2-wk-old 1 .42% NPP
IP6 Group 2 3-wk-old .42% NPP
IPs Group 1 Group 2 2-wk-old 3-wk-old .42% NPP .42% NPP
Group 3 3-wk-old .27% NPP Kul6'b>
'"
Group 3 3-wk-old .27% NPP
•"
34.22 2.7 1.9 nd 27.1 28.1 26.8 4.9" 31.1 30.9 2.8b 2.0b 31.7 4.9* 34.7 36.7 3.3b 2.0c 32.9a 25.4b 25.7* 5.2" 2.8b 2.0c 27.7 5.5" 28.3 26.5 3.3b .9« b 27.8 27.3 29.0 4.1" 3.1 1.2' 32.2 4.3a 27.5 29.4 3.5b 1.6' 3.1 ± .4b 1.6 ± .2' 4.8 ± .2a 30.9 ± 1.1 29.0 ± 1.3 28.7 ± .7 Regression analysis for IF'6 or IP5 concentration in excreta .254 .418
.349 .378
.696 .366
.340 .705
.418 .758
<.001 .001
a_c Means in a row and IP with no common superscripts differ significantly (P £ .05) based on Duncan's multiple range test. !NPP = nonphytate phosphorus. 2 Values are means (n = 3). 3n = 21.
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Effect of Sequential Collection of Excreta from Dropping Trays on Phytate Content
Dietary nonphytate phosphorus
.005
8.0*3 5.7Y 6.5* 6.7 + .2
IPe
.003
.46 ± .04
.6* .6* .3y
IPs
Crop2
.167
3.0 3.8 2.1 3.0 ± .6
IP 6
<.001
.5" .3y .0* .27 ± .04
IPs
Gizzard IPs
.075 <.001
(mg/g) 2.3* 26.9* .5* 19.6X 1.5y 23.2*y 23.2 ± 1.8 1.43 ± .11
IP6
Small intestine
*- Means in a column with no common superscripts differ significantly (P < .05) based on Duncan's m lr The IP5 values were actually calculated from the response to the IP6 standard in the HPLC procedure. The diet con 2 Number of samples were crop n = 2, gizzard n = 3, small intestine n = 3, and cloaca n = 2. The n for the mean+SEM, cro n = 6. 3 Values are means (n = 3).
z
(wk) (%) .42 2 .42 3 .27 3 Means + SEM ANOVA (Probabilities) df Treatmeni t 2
Age of chick
TABLE 4. Quantities of inositol hexaphosphate (IPg) and inositol pentaphosphate (IPs) sections of the gastrointestinal tract (GIT)1
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PHYTATE UTILIZATION IN CHICKENS
1913
IP6
IP
^
fr^N
El-I B IP6
IP4
IP3 17% 6% as
1PS
33
IP6 44%
J" *;
FIGURE 1. Reverse-phase HPLC of inositol phosphates. A) 16.8 /ig of Sigma (Sigma Chemical Co., St. Louis, MO 63178-9916) inositol hexaphosphate (IP6); B) Excreta sample showing peaks for IP6 at 13.2 jig, inositol pentaphosphate (IP5) at 2.7 jig, inositol tetraphosphate (IF4) at 1.3 /*g, and inositol triphosphate (IP3), quantity cannot be calculated; C) The same fecal sample as B but with a known amount of IP6 added as an internal standard, the increase in peak IP6 should equal 5.7 ng and was equivalent to 6.0 ng (105% recovery); D) The Aldrich (Aldrich Chemical Co., Inc., Milwaukee, WI 53233) commercial phytic acid solution. Lehrfeld (1989) reported a similar solution from Aldrich contained IPg, 43%; IP5,38%; IP4,17%; and IPj, 3%. This chromatograph was obtained at a later date than the other three and the retention times were greater.
The calculated retention from crop and gizzard was higher than that from small intestine and cloaca in all three treatment groups. There was a significant difference in the retention of IP6 in crop among the three treatment groups. The retention was highest in Group 3 and lowest in Group 1 (36 versus 25%). Negative IP6 retention values were calculated for the small intestine and cloaca content; and significantly more IP6 was retained by chickens in Group
3 that received the low-phosphorus diet for 3 wk. In this experiment, chromic oxide concentration varied from 1.20 mg/g of feed to .75 mg/g in gizzard digesta, with ash content increasing from 6.36% in feed to 9.18% in gizzard (Table 6). The decrease in chromic oxide concentration and the increase in ash content in the upper portion of the alimentary tract thus resulted in high retention. A build-up of chromic oxide at
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IP3 IP4
1914
SOONCHARERNYING AND EDWARDS, JR. TABLE 5. Calculated retention of inositol hexaphosphate (IP6) from different sections of gastrointestinal tract Retention of IP 6
Intestinal tract organ
Group 1 2-wk-old .42% NPPi
Group 2 3-wk-old .42% NPP
Group 3 3-wk-old .27% NPP
Crop 2 Gizzard Small intestine Cloaca Means ± SEM
25 b 3 56 -11 -8 16.8 ± 8.4
33 a b 45 -22 -16 10.4 ± 9.8
36" 72 7 4 32.0 ± 7.6
S ± SEM
ANOVA probabilities
31.3 57.6 -8.7 -6.7
.049 .124 .174 .477
IO/\
ab ' Means in a row with no common superscripts differ significantly (P £ .05) based on Duncan's multiple range test. WPP = nonphytate phosphorus. 2 Number of samples were crop n = 2, gizzard n = 3, small intestine n = 3, and cloaca n = 2. The n for mean ± SEM for the three groups n = 10, crop n = 6, gizzard n = 9, small intestine n = 9, and cloaca n = 6. 3 Values are means (n = 3).
the posterior portion therefore caused a negative retention. DISCUSSION
The analysis of excreta samples for phytates reported in Table 2 would indicate in general that the amounts of inositol phosphates other than IP5 and IP6 are present in very low amounts and are probably of little significance in most studies. The analysis of excreta phytate by the ferric chloride method gives lower values in the excreta, which would translate into greater phytate retention when it is calculated from these values. Although it is clear that retention values calculated from the ferric chloride method would be too high, the high correlation obtained between the two methods for just eight samples would still indicate that values obtained with the ferric chloride methods are usable for comparative purposes, such as between dietary treatments. In the sequential collection study, great care was taken in collecting and immediately freeze drying the samples. Thus, it is apparent that no significant conversion of IP6 to phosphate and inositol from either enzymatic or microbial action took place in the excreta on the collection pan at room temperature during this 32-h period after defecation. Under the conditions of this experiment, especially the 22 C temperature of the chick room, it is doubtful
that any microbial action after 32 h would cause a significant increase in the loss of phytate, as the droppings are drying at a very rapid rate at this temperature. It is probably acceptable in phytate retention studies to have collection periods of up to 32 h with little problems from phytate breakdown in the droppings. However, if large amounts of phytase are added to a diet or if ingredients are used that contain considerable amounts of phytase, the collection time may need to be reevaluated. Although age and dietary treatment had a significant effect on the retention of IP6/ there was no significant difference in the IP6 content of the excreta. However, there was a significant difference in the IP5 content of the excreta; the 2-wk-old birds having the highest concentration (4.8 mg/ g) followed by the 3-wk-old birds receiving the diet containing .42% nonphytate P (3.1 mg/g) and the 3-wk-old birds receiving the diet containing .27% nonphytate P (1.6 mg/g). The retention figures confirm work snowing that old birds retain more phytate phosphorus (Edwards et al, 1988) and that increasing the phosphorus content of the diet decreases the utilization of phytate phosphorus (Edwards and Veltmann, 1983). The significant effect of age of birds and dietary nonphytate phosphorus on the IP5 concentration in excreta in the sequential collection study indicates the possible
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± 2.3 ± 7.7 ± 9.5 ± 10.2
PHYTATE UTILIZATION IN CHICKENS
1915
TABLE 6. Ash and chromic oxide concentration in feed and ingesta Variable
Feed
Ash Chromic oxide
6.36 ± 1.131 .120 ± .005a
Crop (g/100 g) ± .9 6 .104 ± .007*
6-86
Gizzard 9.18
± ^52 .075 ± .001b
x ± SEM 7.47 ± 2.60 1.00 ± .009
ANOVA probabilities .296 .002
ab
' Means in a row with no common superscripts differ significantly (P £ .05) based on Duncan's multiple range
test. a
Mean ± SEM, n = 3. Group means ± SEM, n = 9.
the chromic oxide technique to determine phytate hydrolysis in the gastrointestinal tract of the golden hamster and the laboratory rat with no evidence that the chromic oxide underwent any kind of unusual distribution. However, high digestibilities of phytate were found for both animals when the diet contained natural phytase. Negative retention of phytate using the chromic oxide method has previously been reported from this laboratory (Elliot and Edwards, 1991a,b). When samples from a large amount of excreta give a negative value, the most likely explanation is that the analysis of the feed was incorrect and that the value was low, probably due to poor extraction. Another explanation of the high retention of IP6 in the crop and gizzard is suggested by the very low levels of IP6 found in the content of these sections of the tract as compared with the feed. For instance, the gizzard content contained only 3.0 m g / g of IP6 compared with 11.4 m g / g of IP6 in the feed. The most apparent explanation of this is that the soybean meal (high in IP6) is constantly moving out of the crop and gizzard but corn (low in IP6) is remaining. In the present studies with chickens, apparent retention of IP6 in different sections of the alimentary tracts using the chromic oxide indicator method is not a reliable value. These results indicate that the chromic oxide indicator method for determining retention is not a reliable method for detecting quantitative disappearance of IP6 in different sections of the gastrointestinal tract. Chromic oxide previously has been found to be unsatisfactory for determining the absorption of individual fatty acids in the different
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advantage that the HPLC method may have over the ferric chloride method in evaluating nutritional effects on phytate utilization. The lower concentrations of IP5 found in the older birds receiving the diet containing .27% nonphytate phosphorus are of particular interest in view of the 44.6% calculated retention value by this group as compared with 32.5% for birds of the same age fed the .42% nonphytate phosphorus. Thus, the high percentage of phytate phosphorus retention is related to a decrease in the concentration of the first breakdown product (IP5) in the excreta. This may be an indication that a key to the amount of phytate utilized is the amount initially broken down to IP5. There was lower recovery of phytate phosphorus from excreta (87.8%) than from contents of the gastrointestinal tract (95.8%). The most likely explanation of the lower than 100% recovery in both type samples is probably that the IP6 became bound to some material in the samples. However, the possibility that hydrolysis is taking place cannot be ruled out from the information that is available at this time. The calculated retention of IP6 in different sections along the alimentary tract resulted from the change in concentration of IP6 and chromic oxide as the digesta passed down the tract. The high retention of IP6 in crop and gizzard may have resulted from disappearance of chromic oxide from these sections. Johnson et al. (1964) found that chromic oxide in wethers was passed more rapidly out of the anterior portion and less rapidly out of the posterior portion of the digestive tract. This resulted in an increase in the concentration of the indicator in the latter portion. Williams and Taylor (1985) used
1916
SOONCHARERNYING AND EDWARDS, JR.
regions of the digestive system in pigs (Carlson and Bayley, 1968) and in rats (Carlson and Bayley, 1972). However, the chromic oxide indicator method still appears to be a feasible method for the determination of digestibility. REFERENCES
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Ashton, W. M , C. Evans, and P. C. Williams, 1960. Phosphorus compounds of oats. II. The utilisation of phytate phosphorus by growing chicks. J. Sci. Food Agric. 11:722-727. Ballam, G. C , T. S. Nelson, and L. S. Kirby, 1984. Effect of fiber and phytate source and of calcium and phosphorus level on phytate hydrolysis in the chick. Poultry Sci. 63:333-338. Brisson, G. J., 1956. On the routine determination of chromic oxide in feces. Can. J. Agric. Sci. 36: 210-211. Carlson, W. E., and H. S. Bayley, 1968. Utilization of fat by young pigs, fatty acid composition of ingesta in different regions of the digestive tract and apparent and corrected digestibilities of corn oil, lard and tallow. Can. J. Anim. Sci. 48: 315-322. Carlson, W. E., and H. S. Bayley, 1972. Preparation and use of a glyceryl triether as an indicator of fat absorption. Br. J. Nutr. 28:295-305. Common, R. H., 1940. The phytic acid content of some poultry feeding stuffs. Analyst 65:79-83. Consortium, 1988. Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. Association Headquarters, Champaign, IL. Edwards, H. M., Jr., 1983. Phosphorus. 1. Effect of breed and strain on utilization of suboptimal levels of phosphorus in the ration. Poultry Sci. 62:77-84. Edwards, H. M , Jr., 1988. Effect of dietary calcium, phosphorus, chloride, and zeolite on the development of tibial dyschondroplasia. Poultry Sci. 67:1436-1446. Edwards, H. M., Jr., and M. B. Gillis, 1959. A chromic oxide balance method for determining phosphate availability. Poultry Sci. 38:569-574. Edwards, H. M., Jr., and J. R. Veltmann, Jr., 1983. The role of calcium and phosphorus in the etiology of tibial dyschondroplasia in young chicks. J. Nutr. 113:1568-1575. Elliot, M. A., and H. M. Edwards, Jr., 1991a. Some effects of dietary aluminum and silicon on broiler chickens. Poultry Sci. 70:1390-1402. Elliot, M. A., and H. M. Edwards, Jr., 1991b. Comparison of the effects of synthetic and natural zeolite on laying hen and broiler chicken performance. Poultry Sci. 70:2115-2130.
Gillis, M. B., K. W. Keane, and R. A. Collins, 1957. Comparative metabolism of phytate and inorganic P 3 2 by chicks and poults. J. Nutr. 62:13-26. Gillis, M. B., L. C. Norris, and G. F. Heuser, 1949. The effect of phytin on the phosphorus requirement of the chick. Poultry Sci. 28:283-288. Graf, E., and F. R. Dintzis, 1982. High-performance liquid chromatographic method for the determination of phytate. Anal. Biochem. 119:413-417. Helwig, J. T., and K. A. Council, 1979. SAS® User's Guide, 1979 ed. SAS Institute Inc., Raleigh, NC. Heuser, G. F., L. C. Norris, J. McGinnis, and M. L. Scott, 1943. Further evidence of the need for supplementing soybean meal chick rations with phosphorus. Poultry Sci. 22:269-270. Johnson, D. E., W. E. Dinersson, and D. W. Bolin, 1964. Rate of passage of chromic oxide and composition of digesta along the alimentary tract of wethers. J. Anim. Sci. 23:499-505. Lehrfeld, J., 1989. High-performance liquid chromatography analysis of phytic acid on a pHstable, macroporous polymer column. Cereal Chem. 66(6)510-515. Lowe, J. T., H. Steenbock, and C. H. Krieger, 1939. Cereals and rickets. IX. The availability of phytin-P to the chick. Poultry Sci. 18:40-44. McGinnis, J., L. C. Norris, and G. F. Heuser, 1944. Poor utilization of phosphorus in cereals and legumes by chicks for bone development. Poultry Sci. 23:157-159. National Research Council, 1985. Guide for the Care and Use of Laboratory Animals. National Institutes of Health Publication No. 85-23, rev. National Institutes of Health, Bethesda, MD. Nelson, T. S., 1967. The utilization of phytate phosphorus by poultry—A review. Poultry Sci. 46:862-871. Oberleas, D., 1971. The determination of phytate and inositol phosphates. Methods Biochem. Anal. 20:87-101. Sandberg, A. S., and R. Ahderinne, 1986. HPLC method for determination of inositol tri-, terra-, penta-, and hexaphosphates in foods and intestinal contents. J. Food Sci. 51:547-550. Sieburth, J. F., J. McGinnis, T. Wahl, and B. A. McLaren, 1952. The availability of the phosphorus in unifine flour for the chick. Poultry Sci. 31: 813-818. Singsen, E. P., and H. H. Mitchell, 1944. Soybean meal chick rations need no inorganic phosphorus supplements. Poultry Sci. 23:152-153. Waldroup, P. W., C. B. Ammerman, and R. H. Harms, 1964. The availability of phytic acid phosphorus for chicks. 2. Comparison of phytin phosphorus sources. Poultry Sci. 43:426-432. Williams, P. J., and T. G. Taylor, 1985. A comparative study of phytate hydrolysis in the gastrointestinal tract of the golden hamster (Mesocricetus auratus) and the laboratory rat. Br. J. Nutr. 54: 429-435.