Phosphorus, but not Calcium, Affects Manganese ...

Minerals and Trace Elements

Phosphorus, but not Calcium, Affects Manganese Absorption and Turnover in Chicks1 KAREN J. WEDEK1ND,2 EVAN C. TITGEMEYER, AND DAVID H. BAKER3

A. ROBERT

TWARDOCK*

Departments of Animal Sciences and *Veterinary Biosciences, University of Illinois, urbana, Â¡L61801

ABSTRACT Two balance studies with growing chicks were conducted to evaluate the effects of excess Ca or excess P on endogenous fecal Mn excretion and true Mn absorption. An isotope-dilution technique was used to estimate endogenous manganese in excreta. Supple ments were added to a corn-soybean diet containing 1% Ca, 0.7% P (0.5% available P) and 37 mg Mn/kg. In Experiment 1, supplemental Ca levels of 0, 0.5 and 1.0% from feedgrade limestone were compared. True absorption of Mn was not affected by Ca level (P > 0.10) and averaged 2.8% for birds fed the Mn-unsupplemented diet. In Experiment 2, a 2 x 3 factorial arrangement of treatments included: 100 and 1000 mg/kg supplemental Mn (from MnSO4-H2O) and 0, 0.4 and 0.8% added P supplied by dicalcium phosphate. Excess P significantly decreased true absorption of Mn (P < 0.01). In birds fed 100 mg/kg supplemental Mn, absorption of Mn decreased 22% as excess P increased from 0 to 0.8%, whereas in birds fed 1000 mg/kg supplemental Mn, Mn absorption decreased 59% as a result of 0.8% P supple mentation. These results confirm that the antagonism of Mn by inorganic P is due to reduced gut absorption of Mn. J. Mutr. 121: 1776-1786, 1991.

MATERIALS AND METHODS Animals, diets and treatments. Housing, Handling and killing procedures conformed with policies and recommendations of the University of Illinois Labo ratory Animal Care Advisory Committee. Male chicks resulting from the cross of New Hampshire males and Columbian females were used in three experiments. All chicks were fed a corn-soybean meal starter diet (168 mg Mn/kg diet) during the first 7 d posthatching. On d 8 posthatching after on overnight fast, the chicks were weighed and allotted randomly to experimental groups. Five replicate groups of four chicks in Experiment 1, or four replicate groups of

INDEXING KEY WORDS:

â€¢manganese â€¢phosphorus â€¢Isotope-dilution â€¢chicks

â€¢calcium

It has been widely accepted in reviews of Mn me tabolism (1-3) that excess Ca and P adversely affect Mn utilization in chicks. Research from our labo ratory (4-6), however, has shown that, whereas P adversely affects Mn utilization, Ca has little or no effect. The criterion used to study the Mnantagonizing effects of excess Ca and P in our earlier studies was the regression of total tibia Mn on supple mental Mn consumed. The results of these studies are summarized in Table 1 (6). Although these bioavailability studies clearly showed that Mn utilization was reduced by excess P, it is not clear to what extent excess P affected absorption or endogenous excretion 0022-3166/91

$3.00 Â©1991 American

Institute

of Nutrition.

'Part of a dissertation

submitted

by Karen J. Wedekind to the

Graduate College, University of Illinois, in partial fulfillment of the requirements for the Ph.D. degree in Animal Sciences. 2Present address: Mark Morris Associates, 5500 SW 7th Street, Topeka, KS 66606. 'To whom reprint requests should be addressed: 328 Mumford Hall, 1301 West Gregory Drive, University 61801.

Received 14 December 1776

1990. Accepted 6 May 1991.

of Illinois, Urbana, IL

Downloaded from jn.nutrition.org by guest on July 10, 2011

of Mn. Earlier studies (7-10) implied that the antago nistic effects of excess Ca or P on Mn were a gut phenomenon because Mn injected intraperitoneally or subcutaneously was more effective in overcoming the growth-depressing effects of excess Ca and P than Mn given in the diet. The primary ami of the present study was to de termine how the absorption and endogenous ex cretion of Mn were affected by excess Ca or P. Isotope-dilution techniques were used to assess en dogenous Mn losses and true absorption of Mn. Briefly, this technique involves the labeling of body Mn after injection of MMn and then measuring the degree of labeling of fecal Mn in relation to that of endogenous pools.

MANGANESE-PHOSPHORUS

INTERACTION

1777

TABLE 1 Manganese utilization

in chicks fed excess caldani

and phosphorus1

added2Mnmg/kg050010001000100010001000Ca%0001.01.001.0p%00000.880.880.88Food

tibia bioavailMnintake3mg0"195d407'349b416'334C418'4.5Total ability*%100.0-91.9-35. intake8401a*391b408lb348C416'333C413'7Supplemental MnMÂ«6.31"11.96"18.33'15.74b10.6

Excess Ca additionNoneNoneNoneCaCOjCaCOj

KHjPO4KHjPO,Dicalcium + phosphatePooled SEMAmount 'Values are means of four pens of three male crossbred chicks per pen during the period 8 to 22 d posthatching. Values with different superscript letters differ (P < 0.05), least significant difference. Data are from rÃ©f.6. 2All mineral supplements were added to the basal corn-soybean meal diet (23% crude protein) at the expense of corn st arch. The basal diet

four chicks in Experiment 2 were fed the experi mental diets from 8 to 24 d posthatching. In Ex periment 3, the individual chick was the experi mental unit, and five chicks were fed the experimental diet from 8 to 24 d posthatching. A corn-soybean meal basal diet (Table 2) was used in all experiments. The diet contained, by analysis, 37 mg Mn/kg, 1.10% Ca and 0.5% available P. Although 37 mg Mn/kg is below the suggested Mn require ments for chicks (60 mg Mn/kg; 11), studies in our laboratory have not shown growth responses to Mn supplementation of this diet (4, 5). Experiment 1 was conducted to assess the effects of excess Ca alone on Mn utilization. Two levels of excess Ca, 0.5% and 1.0%, were compared with a control diet (Table 2) that provided no excess Ca. Excess Ca was supplied as feedgrade limestone at the expense of comstarch. Experiment 2 was conducted to determine the effect of excess P on Mn utilization. Inorganic Mn was provided as MnSO4-H2O at two supplemental levels: 100 and 1000 mg Mn/kg (137 and 1037 mg/kg total Mn, respectively). Three levels of dicalcium phosphate were added to the diet to provide 0, 0.4 and 0.8% excess P (which also provided 0, 0.45 and 0.91% excess Ca, respectively). These combinations of Mn and P were analyzed as a 2 x 3 factorial arrangement of treatments in a completely randomized design. Although dicalcium phosphate provided excesses of both Ca and P, research from our laboratory (cf. Table 1) has indicated that the Mn-antagonistic effect of dicalcium phosphate results from the P and not the Ca it provides. Experiment 3 was conducted to compare the results of 54Mn oral dosing with intramuscular S4Mn

injections. One level of dicalcium phosphate was added to the diet to provide 0.8% excess P and com pared with a control diet (Table 2). Evaluation of manganese excretion and retention. Endogenous excretion, net (or apparent) absorption and true absorption of manganese were computed

TABLE 2 Composition

of basal die?

Ingredient

Amount g/100 g diet

Cornstarch Corn (8.5% crude protein) Soybean meal (48.0% crude protein) Com oil Dicalcium phosphate Limestone Iodized salt DL-Methionine Vitamin premix2 Cholinc chloride Ferric citrate ZnCOj Lincomycin (44 g/kg) Selenium premix3

10.31 39.71 40.89 5.00 2.20 1.00 0.40 0.20 0.10 0.10 0.06 0.01 0.01 0.01 'Contained 23% crude protein, 37 mg Mn/kg, 1.1% Ca and 0.5% available P. 2Vitamin premix provided (per kg diet): retinyl acetate, 1485 ug, cholecalciferol, 25 ug; all-rac-a-tocopheryl acetate, 11 mgÂ¡vitamin B-12, 0.01 mg, riboflavin, 4.41 mg, D-Ca pantothenate, 10.0 mg, niacin, 22.0 mg, menadione sodium bisulfite, 2.23 mg. 'Selenium premix provided 0.1 mg Se/kg diet from Na selenite.


contained 1.1% Ca, 0.51% available phosphorus and 37 mg Mn/kg diet. 'Includes supplemental Mn intake from MnSO4-HjO and from Mn present in the calcium supplements. 'Based upon standard-curve methodology using 0, 500 and 1000 mg Mn/kg to generate a standard curve: Y - 6.2990 + 0.02942X (r - 0.95), where Y - total tibia Mn (ug) and X - supplemental Mn intake (mg).

WEDEKIND

1778

ET AL.

TABLE 3 Equations

used for deriving endogenous excretion, apparent absorption and true absorption from d 9 to d 16 post-uMn injection1

Formula

Symbol

Term Endogenous excretion (d 9-16) Percentage of total excreta Mn m,: d 9-12; nv d 13-16

m, - ^-

Percentage Apparent

(d 9-16)

R(a)

Q, 'Reprinted

R(t)

R(t) - R(a)

rW

,-. _^!v

Specific activity (kBq MMn/mg Mn) of total Mn in excreta collected from d 9 to 12 and d 13 to 16, respectively Specific activity (kBq "Mn/mg Mn) of liver Mn on d 10 and d 14, respectively Intake of dietary Mn from d 9 to 16 |ug Mn/day) Final amount of whole body Mn (ug) after 16 d on treatment Whole body Mn (ug) in representative birds determined at d 0 Liveweight (g) of chicks after 0, 8 and 16 d, respectively with permission

from rÃ©f.14.

according to equations outlined in Table 3. The isotope-dilution method (12-14) was used to estimate the proportion of endogenous manganese in the ex creta. Though excreta in avians contain both feces and urine, little Mn in excreta of chicks is of urinary origin (14). We assumed that the specific activity (SA) of liver manganese 10 and 14 d after ^Mn injection reflected, on average, the SA of endogenous man ganese excreted in feces from d 8 to 12 and d 12 to 16, respectively. It is crucial that a reliable estimate of the SA of endogenous Mn be determined in this tech nique. Among the various tissues and organs ana lyzed, Weigand et al. (13) considered both liver and serum to be suitable sources: the liver because of its key role in Mn homeostasis and its direct contri bution via biliary Mn secretion, and the serum be cause of its transport function, which is responsible for a rapid exchange of Mn among tissues. Of the tissues examined, liver and serum exchanged Mn most rapidly. The isotope-dilution method as well as comparative isotope balance procedures were found to give similar estimates of endogenous fecal excretion (12, 14, 15). Net Mn retention was assessed according to the comparative slaughter technique. Experimental procedures for intramuscular MMn studies. Following the 7-d preliminary period, 518

kBq (Experiment 1) or 370 kBq (Experiment 2) MMnin 0.2 mi. of saune solution was injected intramuscu larly into the right breast muscle. The isotope source was carrier-free ^MnCla in 0.1 mol/L HC1 (Amersham, Arlington Heights, IL). Prior to injection, a representative group of eight chicks was killed for analysis of d-0 whole-body Mn. Experimental diets were offered ad libitum for 16 d after S4Mn injection (d 0). Excreta was collected in successive 4-d periods for each pen (four birds/pen). Dietary intake and weight gain were assessed at suc cessive 4-d intervals. One pen of chicks from each treatment was killed both 10 and 14 d, post-^Mn injection,- the remaining three pens in Experiment 1, and two pens in Experiment 2, were killed at d 16. Each bird was dissected (for separate analysis of stable and radioactive Mn) into liver, tibia and residual body. The gastrointestinal tracts of all birds were removed, split open and rinsed with deionized water to remove gut contents. Carcasses were dipped in scalding water to facilitate removal of feathers, which were discarded. Legs were heated in a pressure-cooker for 30 min to ease removal of muscle from bone. Muscle and skin removed from the tibia and emptied gut tissue were added back to the remaining whole body fraction. Whole carcasses (minus tibia and liver)


c.Â«

.Â«- W.) - W0) x 8

(d 9-16)

Daily rate (jig Mn/day) Percentage of Mn intake

m(I|

1CÂ»-

of Mn intake

True absorption

I

[ml + mj 2 x 100

- y x 100

Daily rate (ug Mn/day) Percentage

m, - ^=- x 100 Smffl

E* - (I - R(a() x

of Mn intake

absorption

x 100

Sm(l|

Daily rate (ug Mn/day)

of manganese in chicks

MANGANESE-PHOSPHORUS INTERACTION

3, 4, 5, 6, 8, 12, 14 and 16 d post-dosing. StatÃ¬stica!analyses. Data from the balance study were analyzed by ANOVA for a completely ran domized design using the general linear model (GLM) procedure of SAS (16). Treatment means were com pared using single degree of freedom orthogonal con trasts (17). Data from the whole-body counter were analyzed using the nonlinear regression procedure (3PR) of BMDP Statistical Software (18), with the natural logarithm of percent "Mn dose regressed over time. A comparison was made between two, three or four parameter models, and the four-parameter model was found to fit the data best as indicated by the lowest residual mean square, and expressed by the equation: Y -

+ a2eblt

In a four-parameter model, both a slow and fast pool are resolved. The exponents of the regression equa tions, bj and bj, specify the fractional turnover rates. Half-life estimates were determined from the frac tional turnover rates as (hi 0.5/b). The coefficients 3j and &2represent the percentage of the total Mn pools that could be resolved respectively, into either slow or fast turnover times.

RESULTS Determination of endogenous Mn and true Mn absorption. The observed mean values for the SA of excreta and liver are reported in Table 4. No effects of Ca were observed for either liver or excreta SA (P >

TABLE 4 Endogenous

Ma in excreta on d 8 to 16 following "Mn injection in chicks as determined by ratio of the specific activity excreta and liver: effect of excess dietary Ca (Experiment If

of Mn in

Ca0.5%11.43 EM.85

Specific activity, kBq uMn/mg Excreta (d 8-12) Excreta (d 12-16) Liver (d 10) 14)Endogenous Liver (d

Mn 7.77 434.75 321.902.50

7.96 592.00 249.751.93

7.22 518.00 271.952.39

1.150.17

3 1 14

Mn in excreta, % of total Mn 8-12)m'' Excreta |d liver (d 10) 12-16)â„¢2' Excreta |d liver (d 14)0%10.88

2.41Excess 3.181.0%12.40 2.65S 0.42jr14 3 'The basal diet for this experiment contained 37 mg Mn/kg, 1.1% Ca and 0.5% available P; excess Ca was provided as CaCO3. 2The number of replicates involved in calculating the means are indicated by n. Although there is no true replication for liver specific activity determinations, livers from the two heaviest and two lightest birds within pens were pooled. Within these two subsamples, duplicate determinations were made.


were freeze-dried; remaining tissues were oven-dried for 24-48 h at 105'C. Excreta and tibia were dryashed (600"C) in a muffle-furnace, followed by wetashing with HNO3, or both HNO3 and H^Oz in the case of tibia. Liver was wet-ashed with HNO3/ and a combination of HNO3 and HfÃ¬y. was used to wet-ash residual body. The solutions that resulted from wetashing were analyzed for Mn concentration via atomic absorption spectrophotometry (Perkin-Elmer, Model 306, Norwalk, CT) and "Mn activity by gamma scintillation spectrometry [TM Analytic Model 1185, changer and detector; Canberra Model 8100 multichannel analyzer; well-type detector: Nal (TI) crystal]. Tissue count rates were measured simul taneously with "Mn standards prepared from the dose solutions and corrected for background. Results were expressed as kilobecquerels of 54Mnper gram of tissue or as the percentage of administered dose. Whole-body "Mn retention over the course of the 16-d period was assessed at 1, 2, 3, 4, 5, 6, 8, 12 and 16 d post-dosing using a dual Na (Tl) crystal whole-body counter connected to a multichannel analyzer. The live birds were group-counted and compared with an equivalent dose of "Mn diluted in a 100-mL volu metric flask and counted in the same counting geom etry. Experimental procedures for oral MMn dose study. Following the 7-d preliminary period, 1.67 MBq of "Mn, contained in 1 mL of sahne solution, was cropintubated into each bird. Experimental diets were offered ad libitum for 16 d after "Mn oral-dosing (d 0). Whole-body "Mn retention over the course of the 16-d period was assessed using procedures similar to the intramuscular "Mn studies, with the exception that the live birds were individually counted at 1, 2,

1779

WEDEKIND

1780

ET AL.

TABLE 5 Endogenous fecal excretion,

and apparent

and true absorption of Mn in chicks in response to excess dietary Ca during d 8 to 16 of the balance study (Experiment 1)' Ca0.5%1215.230.37

Mn ng/cPEndogenous intake, Mn excretion, VÂ£/d intakeApparent % of absorption,VX/d intake*True % of

2.353.72

2.503.01

2.524.49

0.210.61

0.26337.00

0.25033.38

0.41631.65

0.0463.14

absorption,Hg/d % of intake0%1414.933.28

0.10). The percentage of total Mn in excreta that was of endogenous origin was not affected by Ca level {P> 0.10) and averaged 2.5%. Table 5 shows the results during d 8 to d 16 of the balance period. There was a significant linear decrease in dietary Mn consumed as a result of increased Ca intake (P < 0.05). This occurred because the limestone added to the diet to achieve the levels of excess Ca reduced voluntary food intake of the chicks. The daily fecal excretion of endogenous Mn averaged 30.3 ng/d and was not affected by Ca level. Although Mn intake differed among treatments, endogenous Mn losses ex pressed as a percentage of Mn intake did not differ (P > 0.10). Apparent absorption (net retention) of Mn (ug/ d) was not affected by Ca level (P > 0.10). Apparent absorption of Mn expressed as a percentage of intake, however, was slightly elevated in birds fed 1% excess Ca compared with those fed lower Ca levels. True absorption of Mn, whether expressed as micrograms per day or as percentage of intake, was not affected by Ca level (P > 0.10). The percentage of Mn truly ab sorbed averaged 2.8%. The SA of excreta for each 4-d period increased with P supplementation for birds fed the 100 mg/kg supplemental Mn but not for birds fed 1000 mg/kg supplemental Mn (Table 6). Thus, a Mn x P inter action (P < 0.01) existed for excreta SA. Increases in liver SA occurred with increasing P addition at both levels of Mn intake. As expected, liver and excreta SA were lower for birds fed higher Mn levels. In general, the percentage of total excreta Mn that was of endog enous origin decreased as dietary excesses of P in creased. The decrease in the contribution of endog enous Mn to total Mn excretion was greater for birds

fed 1000 mg/kg supplemental Mn than for those fed 100 mg/kg supplemental Mn [Mn x P interaction (P < 0.01)]. Differences noted in the liver SA data sug gested that the absorption of dietary Mn was reduced in the presence of excess P. A significantly reduced absorption of stable Mn was counteracted by a decrease in turnover or excretion of ^Mn, thereby yielding a higher SA. Thus, homeostatic mechanisms compensated for the decreased absorption of Mn by increasing whole-body Mn retention. The effects of excess P on Mn utilization are sum marized in Table 7. Interactions between Mn and P were present for endogenous fecal Mn excretion, whether expressed as a rate (P < 0.01) or as a per centage of intake (P < 0.05). When birds were fed 100 mg/kg supplemental Mn, only the highest addition of P decreased endogenous Mn excretion. In birds fed 1000 mg/kg supplemental Mn, a linear decrease in endogenous Mn excretion occurred with increasing P supplementation. Apparent absorption (jig/d) of Mn was not affected by P addition (P > 0.10), but was affected by Mn level. As supplemental Mn increased from 100 to 1000 mg/kg, the apparent absorption of Mn (percentage of intake) decreased 67%, falling from 0.65 to 0.21% (P < 0.01). In accord with the responses seen for endogenous Mn excretion, a Mn x P inter action was also noted for true Mn absorption. In birds fed 100 mg/kg supplemental Mn, only the highest addition of P decreased true absorption, whereas birds fed 1000 mg/kg supplemental Mn demonstrated a linear decrease in true Mn absorption in response to increased P. Retention of whole-body **Mn. There was little effect of excess Ca on MMn retention in Experiment 1


2.62Excess 2.751.0%1078.027.16 2.94SEM65.32.80 0.23 'Values are means of three pens of four male chicks/pen during the period 8-16 d post-^Mn injection. The basal diet for this experiment contained 37 mg Mn/kg, 1.1% Ca and 0.5% available P; excess Ca was provided as CaCO3. ^Linear effect of Ca [P < 0.05) as assessed by orthogonal contrasts. 31% excess Ca different from 0 and 0.5% excess Ca (P < 0.05} as assessed by orthogonal contrasts.


INTERACTION

1781

TABLE 6 Endogenous Mn in excreta of chicks on d 8 to 16 following

Excess P (%): SupplementalMn(mg/kg):0 1000.41000.81000 Specific activity, kBq MMn/mg Mn Excreta (d 8-12)3-4 9.92 5.07 Excreta (d 12-16)3-* 2.29 4.07 Liver |d 10) 72.67 107.26 Liver (d 14) 27.53 58.57 Endogenous Mn in excreta, % of total Mn m':

Excreta (d 8-12)3 liver (d 10) Excreta (d 12-16)3

6.98

9.24

"Mn infection: effect of excess dietary P (Experiment

n1

10000.410000.81000SEM 9.18 4.00 136.75 73.11

0.81 0.41 13.91 5.14

0.81 0.44 17.50 9.06

31.12 10.95

6.71

5.85

4.65

2.50

0.78 0.37

0.50 0.04

0.51

3

(Table 8). In Experiment 2, however, increasing di etary excesses of P tended to increase that proportion of the Mn pools that turned over slowly, but did not greatly affect the turnover rates per se. For example, the chicks fed 100 mg/kg supplemental Mn with no excess P retained -40% of the 54Mndose with a mean half-life of 4.3 d, and -60% of the dose with a half-life of 0.66 d. Chicks fed 100 mg/kg supplemental Mn + 0.8% excess P retained 60% of the dose in their slowreleasing pools with a half-Ufe of 4.5 d, and -40% of the dose with a half-life of 0.71 d. Turnover of body manganese was greatly increased by increasing Mn level (Table 8). Birds fed the Mnunsupplemented diet without excess Ca in Ex periment 1 retained -59% of the 54Mn dose in slowreleasing pools with a half-Ufe of 8.5 d, whereas birds fed diets with no excess P and 100 or 1000 mg/kg supplemental Mn (Experiment 2) retained approxi mately 40 and 26% of the "Mn dose, respectively, in pools with long half-lives averaging 4.5 d. Varying Mn intake thus affected both the proportion of Mn pools that turned over slowly as well as the biological halflife of that pool. The data from the oral 54Mnstudy (Fig. 1 and Table 9) indicated that in birds fed 0 and 0.8% excess P, 93 and 98% of the MMn dose, respectively, was con tained in the fast-turnover pools. These fast pools presumably reflect that MMn excreted without being absorbed. The amount of 54Mncontained in the slow pool (aj represents that portion truly absorbed. The absorption of MMn decreased from 6.7 to 2.1% as 0.8% excess P was added to the diet. For the Mn absorbed, birds fed diets containing no excess P had a slow-pool half-Ufe of 2.7 d compared with a half-Ufe of 5 d for birds fed 0.8% excess P.

Manganese concentrations and 54MnrÃ©tentionof various tissues. There were no effects of excess Ca on either stable or ^Mn deposition in various tissues of chicks fed diets containing 37 mg Mn/kg (Experiment 1). Averaged across excess Ca levels, Uver, tibia and whole body contained 35.0, 9.8 and 118 ug of Mn, respectively, at d 16 of the experiment; ^Mn (per centage of dose injected) values were 3.1% for Uver, 1.9% for tibia and 17.3% for whole body. Liver and tibia Mn concentrations were decreased by excess P, but whole body Mn concentration was not significantly affected (Table 10). Increasing levels of P resulted in a linear decrease in Uver and tibia Mn concentration (P < 0.01). There also was a small, but significant increase in Uver Mn concentration (P < 0.001) in response to the 10-fold increase in Mn in take. The total content of Mn in Uver was not af fected by P level (P > 0.10) but was affected by Mn intake (P < 0.001). Manganese content of tibia (micrograms) decreased linearly (P < 0.01) as P level in creased. The percentage of MMnretained in Uver, tibia and whole body increased linearly in response to excess P addition at both levels of Mn supplementa tion. The retention of MMn in all tissues examined was two to three times greater in birds fed 100 mg/kg supplemental Mn compared with birds fed 1000 mg/ kg supplemental Mn. That the retention of MMn in creased as a result of excess P consumption suggests that excess P decreased absorption of stable Mn. The body compensated for decreased Mn absorption by decreasing whole-body Mn turnover. This homeostatic mechanism was confirmed by the wholebody counting data.


8.33 6.95 5.47 7.91 4.90 liver |d 14) 3.38 0.16 2 'The basal diet for this experiment contained 37 mg Mn/kg, 1.1% Ca and 0.5% available P; excess P was provided as CaHPO4. 2The number of replicates involved in calculating the means are indicated by n. Although there is no true replication for liver specific activity determinations, livers from the two heaviest and two lightest birds within pens were pooled. Within these two subsamples, duplicate determinations were made. 3Mn x P interaction (P < 0.01), ANOVA. 'Main effect due to Mn (P < 0.01), ANOVA.

WEDEKIND ET AL.

1782

TABLE 7 Endogenous fecal excretion, and apparent

and true absorption of Mn in chicks determined effect of excess dietary P (Experiment 2)'

Excess P (%): SupplementalMn(mg/kg):0 Mn intake, (ig/d1

1000.4 5352

1000.8 5465

1000 5497

10000.4 40,443

during d 8 to 16 of the balance study:

10000.8 39,584

1000SEM 43,246

424

Endogenous Mn excretion, Â«/Â«P% intake1Apparent of absorption,ug/d4% intake*True of absorption,ug/d2%

of intake1404.87.5735.00.65439.88.22422.17.7236.10.66458.28.38317.55.7835.40.64352.96.422786.06.8991.70.232877.77.121808.54.5783.10.211891.7

DISCUSSION This investigation indicates that excess dietary Ca has no deleterious effect on Mn metabolism. The lack of effect of Ca on the absorption and endogenous excretion of Mn (Tables 4 and 5) was verified by results of whole-body counting (Table 8) as well as data on tissue Mn concentrations. These observations conflict with the commonly held view concerning existence of a Ca-Mn antagonism (1-3). The only adverse effect of excess Ca noted in our study was that excess Ca reduced food intake, thereby reducing Mn intake. Early work in chicks (19-22) that reported Ca-Mn antagonisms used the incidence or severity of perosis as their criterion for assessing Mn antagonism. The increased incidence of perosis observed with in gredients such as steamed bone meal and dicalcium phosphate was often attributed to Ca, despite the presence of P in these products. These studies also failed to report food intake. For an antagonism to exist, the decreased performance must not be merely an effect of decreased food intake. Thus, an important consideration in studying the antagonisms between Ca, P and Mn is the Ca:P ratio in the diet. Previous work in our laboratory (5) had indicated that when the Ca:(available) P ratio falls outside the range of 1.7-3.5, food intake is markedly depressed. The ad dition of either 1% excess Ca or 0.88% excess P, alone, to a corn-soybean meal diet results in decreased food consumption (Table 1). This is espe cially true for the addition of 0.88% excess P. The addition of 1% excess Ca and 0.88% excess P together (i.e., that amount provided from dicalcium phosphate) does not depress food intake. Data in Table 1 indicate

that the Mn-antagonistic effect of dicalcium phos phate is attributable to the P content and not the Ca. Dicalcium phosphate was therefore chosen as the P source in our radioisÃ³topo study, because it provided the level of P desired without depressing food intake. Differential intakes of experimental diets can con found the interpretation of mineral bioavailability and metabolism studies. Because radioisotopic analysis is more sensitive than other procedures, it was important to minimize food intake differences by maintaining an optimal Ca:P ratio. Excess P markedly decreases Mn utilization. This antagonism was evident from the effects of P on endogenous Mn excretion and true Mn absorption (indicated in Tables 6 and 7) and was further substan tiated by whole-body counting (Tables 8 and 9, Fig. 1) and tissue Mn concentrations (Tables 1 and 10). Exis tence of a P-Mn antagonism may be of concern not only in poultry nutrition but in human nutrition as well. The current recommended dietary allowance for P is 800 mg for adult men and women (23). The average daily intake of P in adults, however, is of the order of 1500-1600 mg (24, 25). Not only is P con sumed in excess, but Mn intakes in the American diet are often marginal (26, 27), thereby increasing the likelihood for Mn deficiency. Strause and Saltman (28) suggested that Mn deficiency may be associated with osteoporosis. Both P excesses (29, 30) and Mn deficiencies (28) have been shown to increase bone rÃ©sorptionlosses. Thus, antagonism of Mn by excess P ingestion may exacerbate bone-loss diseases such as osteoporosis. In earlier studies conducted in our laboratory (5), the Mn-P antagonism was studied using a slope-ratio


'Values are means of two pens of four male chicks/pen during the period 9-16 d post-MMn injection. The basal diet for the experiment contained 37 mg Mn/kg, 1.1% Ca and 0.5% available P; excess P was provided as CaHPO4. 2Mn x P interaction (P < 0.01), ANOVA. 3Mn x P interaction Â¡P< 0.05), ANOVA. 'Main effect due to Mn (P < 0.01), ANOVA


INTERACTION

1783

TABLE 8 Calculation

Expt.1221

of half-life of Mn upon whole body "Mn retention in chicks following

injection1*

(d"1)3bi-0.082-0.098-O.098-0.161-0.150-0.154-0.149-0.164-0.158-0.082-0.161-0 distribution3*i%59.366.965.139.655.260.525.726.732.459.339.625.7a*of Suppl. P(mg/kg) Mn Ca or (%)00010010010010001000100001001000none0.5% mean square0.8850.7700.6300.6720.7390.5770.2750.5 pool8.457.077.074.304.624.504.654.2 poold0.870.620.770.660. dose40.733.134.960.344.739.574.473.267.640.760.374.4Exponent

Ca1% Canone0.4% P0.8% Pnone0.4% P0.8% PnonenonenoneFractional Ã„.2Excess

the (3PR) procedure of BMDP. 'Values within a column at a common Mn level (or across Mn levels for Experiments 1 and 2 combined) with unlike superscript letters differ (P < 0.05) as determined by paired t tests for populations with unequal variances. 5Half-life - (hi 0.5)/b.

technique to assess Mn bioavailability (ie., regression of bone Mn content on Mn consumed). The results of this study indicated 0.4 and 0.8% excess P decreased Mn bioavailability by 22 and 38%, respectively. Al though dietary Mn utilization decreased in the presence of excess P, it was not known whether excess P decreased absorption of Mn, increased endog enous Mn excretion, or both. With the use of ^Mn, however, the effects of P on these processes could be quantified. As indicated in Table 7, there was a Mn x P interaction noted for both endogenous fecal Mn excretion and true absorption of Mn. Endogenous fecal Mn excretion (in micrograms per day) expressed as a percentage of true absorption rate, however, did not change with respect to P level. This implies that the Mn excretion from endogenous pools was only affected by P as a result of P decreasing Mn absorp tion. The decrease in endogenous fecal Mn in re sponse to P observed in birds fed 1000 mg/kg supple mental Mn and the decrease in endogenous Mn noted at the highest level of P in birds fed 100 mg/kg supplemental Mn probably occurred because of the concurrent decrease in Mn absorption. Likewise, if excess P were to increase endogenous Mn excretion, this would be expected to decrease apparent Mn ab sorption rate. Nether apparent Mn absorption (micrograms per day) nor endogenous Mn excretion (micrograms per day) expressed as a percentage of truly

absorbed Mn were affected by P level. A change in Mn absorption is unlikely to occur without a con comitant change in endogenous Mn excretion, making it difficult to distinguish whether P decreases endogenous Mn excretion only as a result of decreasing Mn absorption or whether P affects Mn excretion per se. A turnover study (31), however, has shown that excess P has little effect on Mn excretion, thus supporting our contention that excess P affects Mn absorption and not Mn excretion. Although results of both the oral MMn study and the intramuscular MMn study indicated that excess P decreased true absorption of Mn, the estimates of absorption obtained from these two studies did not agree closely. For example, both intramuscularly in jected and crop-intubated birds were fed Mnunsupplemented corn-soybean meal diets in the ab sence of excess Ca or P. The true Mn absorption in the intramuscularly injected MMn-dosed birds was estimated to be 2.6% (Table 5) vs. 6.7% for birds orally dosed with MMn (Table 9). The results of radioisotope-dilution studies gave estimates similar to those obtained using the comparative balance pro cedure (12, 14, 15); the absorption estimates obtained from studies using only orally dosed birds probably overestimated Mn absorption. Based upon the differences in Mn intake between Experiments 1 and 2, one might have expected that true absorption of Mn would be higher in birds fed


'The basal diet in this experiment contained 37 mg Mn/kg, 1.1% Ca and 0.5% available P. *Fivepens of chicks (Experiment 1)and four pens (Experiment 2) were group-counted at 0, 1, 2,3, 4, 5, 6, 8, 12 and 16 d post-MMn injection. Because some pens were killed at d 10 and d 14, only four and three pens (Experiment 1)and three and two pens (Experiment 2) were counted on d 12 and d 16, respectively. 'Percent whole body **Mnregressed on time (d)using the equation: Y - a,eklt + a^'. Nonlinear regression analysis was performed using

WEDEKIND ET AL.

1784

TABLE 9 Calculation

of half-life of Ain based upon whole body "Mn retention following

"Mn oral-dosing

of chicles (Experiment 3)'-2

meansquare0.1000.027Half-life4'51st pool2.75.02nd poold0.2940.35fc

p00.8FractionalÂ«i%6.73'2.09bdistribution3-4a*of (mg/kg)00Excess

dose93.27'97.90bExponentb:-0.0106-0.0058Wb,-0.0998-0.0817Residual

'The basal diet in this experiment contained 37 mg Mn/kg, 1.1% Ca and 0.5% available P; excess P was provided as CaHPOÂ«. 2Five chicks/treatment were individually counted at 0, 1, 2, 3, 4, 5, 6, 8, 12, 14 and 16 d post-MMn oral dosing. ^Percent whole body MMn regressed on time (h) using the equation: Y - a^' + a^e . Nonlinear regression analysis was performed using the (3PR) procedure of BMDP. "Values within a column with unlike superscript

letters differ (P < 0.05) as determined

by paired t tests for populations

with unequal

variances. 5Half-Ufe - (hi 0.5)/b.

kg from the basal corn-soybean meal diet and 100 mg Mn/kg from supplemental MnSO4-H2O, it is possible to estimate the true absorption of Mn from the basal diet as well as that from the MnSOvHjO supplement. Data in Experiment 1 (no excess Ca) indicated that the 37 mg Mn/kg in the corn-soybean meal diet was absorbed at an efficiency of 2.62%. Data in Ex periment 2 (no excess P) indicated that the 137 mg Mn/kg (37 mg/kg from basal, 100 mg/kg from MnSCVHiO) was absorbed at an efficiency of 8.22%. Use of data in Tables 5 and 7 allows an estimate that the 100 mg Mn/kg supplemented as MnSCVHLjOwas absorbed at an efficiency of 10.3%.


Mn-unsupplemented diets than for those fed 100 mg/ kg supplemental Mn. The poor absorption rate of 2.62% noted in Experiment 1 indicates that the Mn contained in com and soybean meal is poorly avail able. Other researchers (32-35) reported that compo nents contained in com and soybean meal markedly reduce Mn bioavailability. These food ingredients not only contain poorly available Mn, but they also reduce the bioavailability of inorganic Mn supple ments. Broiler diets in practice generally contain -137 mg Mn/kg, similar to one of the levels of Mn studied in Experiment 2. Because this diet contained 37 mg Mn/

TABLE10 Manganese content of tissues and "Mn retention in chicks as affected by excess P and varied Mn intake determined

on d 16 of

the balance study (Experiment 2)' Excess P (%): Supplementary Mn (mg/kg):Liver

10021.047.40.217.913.40.464.6504.96.240.410016.243.50.365.59.80.684.5517.49.440.8 10016.842.70.43498.30.724.7495.010.430 100023.155.40.06618.029.40.2010.81196.63.600.4100022.155.40.07011.318 100020.450.90.138.417.70.4011.51272.84.4

Mn,us/Â«2-3MS2"Mn, dose1'3Tibia % o/ Mn,US/Â«213Wi2"3"Mn, dose2-3Whole % o/ Mn,4M&/S2W?2"Ain, body

% of dose1'30 'Values are means of two pens of four male chicks/pen determined on d 16 of the study. The basal diet for the experiment Mn/kg, 1.1% Ca and 0.5% available P. All determinations are expressed on a dry matter basis. 2Main effect due to Mn (P < 0.001), ANOVA. 3P linear effect (P < 0.01) as assessed by orthogonal contrasts. 4Whole body Mn content minus liver and tibia.

contained 37 mg

MANGANESE-PHOSPHORUS 10

g O â€¢o 8

o

7

#

6

o

84Mn dose

5

â€¢*-Â»

4

â€¢*-â€¢ 0)

3

C

Oral

2 1 O O

2

4

6

8

10

12

14

16

18

Days after "Mn injection

In conclusion, the results of the present study provide quantitative evidence that excess Ca has no effect on Mn utilization in the chick. Manganese utilization, however, is reduced by excess P, and this results from decreased gut absorption of Mn. Other work from our laboratory (4, 5) has provided clear evidence that the antagonism of Mn by P is inde pendent of Ca intake. Thus, P salts not containing Ca have been found as antagonistic to Mn utilization as those (such as dicalcium phosphate) that contain both Ca and P.

LITERATURE CITED 1. Leach, R. M. &. Lilburn, M. S. (1978) Manganese metabolism and its function. World Rev. Nutr. Diet. 32: 123-134. 2. Underwood, E. J. (1981) Manganese. In: The Mineral Nutrition of Livestock, 2nd Ã©d.,pp. 125-131. Commonwealth Agricul tural Bureaux, Slough, U.K. 3. Hurley, L. S. &. Keen, C. L. (1987) Manganese. In: Trace Elements in Human and Animal Nutrition, 5th ed. (Mertz, W., ed.), pp. 185-223. Academic Press, San Diego, CA. 4. Wedekind, K. J. Ã„.Baker, D. H. (1990) Manganese utilization in chicks as affected by excess calcium and phosphorus ingestion. Poult. Sci. 69: 977-984. 5. Wedekind, K. J. & Baker, D. H. (1990) Effect of varying calcium and phosphorus level on manganese utilization. Poult. Sci. 69: 1156-1164. 6. Wedekind, K. J. (1990) Manganese Utilization as Affected by Excess Calcium and Phosphorus. Ph.D. Dissertation, Uni versity of Illinois, Urbana, IL. 7. Lyons, M., Insko, W. M. Jr., & Martin, J. H. (1938) The effect of intraperitoneal injections of manganese, zinc, aluminum and iron salts on the occurrence of slipped tendons in chicks. Poult. Sci. 17: 12-16. 8. Wiese, A. C., Johnson, B. C., Elvehjem, C. A. & Hart, E. B. (1938) Phosphorus metabolism of chicks affected with perosis. Science (Washington, DC) 88: 383-384.

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9. Casky, C. D. & Norris, L. C. (1939) Relative effectiveness of ingested and injected manganese in preventing perosis. Proc. Soc. Exp. Biol. Med. 40: 590-593. 10. Schaible, P. J. &. Bandemer, S. L. (1942) The effect of mineral supplements on the availability of manganese. Poult. Sci. 21: 8-14. 11. National Research Council (1984) Nutrient Requirements of Poultry, 8th ed. National Academy Press, Washington, DC. 12. Comar, C. L., Monoe, R. A., Visek, W. J. &. Hansard, S. L. (1953) Comparison of two isotope methods for determination of endogenous fecal calcium. J. Nutr. 50: 459-467. 13. Weigand, E., Kirchgessner, M. &. Heibig, U. (1986) True ab sorption and endogenous fecal excretion of manganese in re lation to its dietary supply in growing rats. Biol. Trace Elem. Res. 10: 265-279. 14. Weigand, E. &. Kirchgessner, M. (1988) Endogenous excretion and true retention of manganese in response to graded levels of dietary Mn supply in chicks. J. Anim. Physiol. Anim. Nutr. 60: 197-208. 15. Weigand, E. & Kirchgessner, M. (1985) Radioisotope studies on true absorption of manganese. In: Trace Element Metabolism in Man and Animals (TEMA-5) (Mills, C. F., Bremner, L &. Chester, J. K., eds.), pp. 506-509, Commonwealth Agricultural Bureaux, Slough, U.K. 16. SAS Institute Inc. (1982) SAS User's Guide: Statistics. SAS Institute Inc., Cary, NC. 17. Steel, R.G.D. &. Tome, J. D. (1980) Principles and Procedures of Statistics: A Biometrical Approach, 2nd ed. McGraw-Hill, New York, NY. 18. BMDP (1979) BMDP Statistical Software, Inc. Los Angeles, CA. 19. Payne, L. F., Hughes, J. S. &. Leinhardt, H. F. (1932) The etiological factors involved in the malformation of bones in young chickens. Poult. Sci. 11: 158-165. 20. Wilgus, H. S., Jr., Norris, L. C. A. Heuser, G. F. (1937) The effect of various calcium and phosphorus salts on the severity of perosis. Poult. Sci. 16: 232-237. 21. Wiese, A. C., Elvehjem, C. A. &. Hart, E. B. (1938) Studies of the prevention of perosis in the chick. Poult. Sci. 18: 33-37. 22. Wilgus, H. S., Jr. &. Patton, A. R. (1939) Factors affecting manganese utilization in the chicken. J. Nutr. 18: 35-45. 23. National Research Council (1980) Recommended Dietary Al lowances, 9th ed. National Academy of Sciences, Washington, DC. 24. Food and Drug Administration (1975) In: Compliance Program Evaluation. FY74 Selected Minerals in Foods Survey, Program circular 7320.08c, p. 6. U.S. Department of Health, Education and Welfare [Health and Human Services], Washington, DC. 25. Page, L. & Friend, B. (1978) The changing United States diet. BioScience 28: 192-197. 26. Greger, J. L., Davis, C. D., Suttie, J. W. & Lyle, B. J. (1990) Intake, serum concentrations, and urinary excretion of man ganese by adult males. Am. J. Clin. Nutr. 51: 457-461. 27. Freeland-Graves, J. H., Behmardi, F., Bales, C. W., Dougherty, V., Lin, P.-H., Crosby, J. B. &. Trickett, P. C. (1988) Metabolic balance of manganese in young men consuming diets con taining five levels of dietary manganai- j. Nutr. 118: 764â€”773. 28. Strause, L. &. Saltman, P. (1987) Role of manganese in bone metabolism. In: Nutritional Bioavailability of Manganese (Kies, C., ed.), pp. 46-55, American Chemical Society Sym posium Series 354. ACS Publishing, Washington, DC. 29. Draper, H. H., Sie, T. L. &.Bergen, J. G. (1972) Osteoporosis in aging rats induced by high phosphorus diets. J. Nutr. 102: 1133-1142. 30. Bell, R. R., Draper, H. H., Tzeng, D.Y.M., Shin, H. K. & Schmidt, G. R. (1977) Physiological responses of human adults to foods containing phosphate additives. J. Nutr. 107: 42-50. 31. Wedekind, K. J., Murphy, M. R. &. Baker, D. H. (1991) Man ganese turnover in chicks as affected by excess phosphorus


FIGURE 1 Percent S4Mn retention in whole body regressed on time following 54Mn oral-dosing in birds fed either 0 (-P) or 0.8% (+P) excess P (Experiment 3). Points represent observed mean values. Coefficients determined from the regression equations fitted for each treatment are presented in Table 9.

INTERACTION

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WEDEKIND ET AL.

consumption. J. Nutr. 121: 1035-1041. 32. Ilalpin, K. M. & Baker, D. H. (1986) Manganese utilization in the chick: effects of corn, soybean meal, fish meal, wheat bran and rice bran on tissue uptake of manganese. Poult. Sci. 65: 995-1003. 33. Halpin, K. M. &. Baker, D. H. (1986) Long-term effects of com, soybean meal, wheat bran and fish meal on manganese util

ization in the chick. Poult. Sci. 65: 1371-1374. 34. Halpin, K. M. &. Baker, D. H. (1987) Mechanism of the tissue manganese-lowering effect of com, soybean meal, fish meal, wheat bran and rice bran. Poult. Sci, 66: 332-^340. 35. Davis, P. N., Noms, L. C. &. KratzÂ«, F. H. (1962) Interference of soybean proteins with the utilization of trace minerals. J. Nutr. 77: 217-223.
