(c)Copyright 1986 by Tile Humana Press Inc. All rights of any nature whatsoever reserved. 016.'~4984186110(M-0265503.00

True Absorption and Endogenous Fecal Excretion of Manganese in Relation to Its Dietary Supply in Growing Rats E. WEIGAND,t M. KIRCHGESSNER,* AND URSULA HELBIG lnstitut fhr Em~hrungsphysiologie, Technische Universit~t /VlOnchen, D-8050 Freising-Weihenstephan, Federal Republic of Germany Received January 26, 1986; Accepted February 28, 1986

ABSTRACT A conventional balance study with 48 male weanling rats was conducted to determine true absorption and endogenous fecal excretion of manganese (Mn) in relation to dietary Mn supply, following the procedures of a previously adapted isotope dilution technique. After 10 d on a diet with 1.5 ppm Mn, eight animals each were assigned to diets containing 1.5, 4.5, 11.2, 35, 65, or 100 ppm Mn on a dry-matter basis. Three days later, each rat was given an intramuscular ~ M n injection and kept on treatment for a balance period of 16 d. Apparent Mn absorption assessed for the final 8 d, averaged 8.6 i~g/d without significant treatment effects, although Mn intake ranged from 18.6 to 1200 p,g/d, in direct relation to dietary Mn concentrations. Mean fecal excretion of endogenous Mn for the six treatments was 0.9, 2.7, 7.4, 11.0, 16.3, and 17.7 #g/d, respectively. These values delineate the rates to which true absorption exceeded apparent rates. True absorption, as percent of Mn intake, averaged 28.7, 15.9, 11.7, 6.1, 3.4, and 2.0, respectively, as compared with mean values of 23.9, 10.9, 6.2, 3.4, 1.2, and 0.5 for percent apparent absorption. It was concluded that both true absorption and endogenous fecal excretion markedly responded to Mn nutrition and that the reduction in the efficiency of true absorption was quantitatively the most significant homeostatic response for maintaining stable Mn concentrations in body tissues. *Author to whom all correspondence and reprint requests should be addressed. tPresent affiliation: Institut for Tierernahrung, Oustus Liebig-Universitat, D-6300 Giessen, F.R.G. Biological Trace Element Research

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Index Entries: Absorption, of manganese in rats, true and apparent; excretion, of endogenous manganese; excretion, of radiomanganese; homeostasis, of manganese metabolism; manganese, true and apparent absorption in rats; manganese, endogenous excretion; manganese, tissue concentrations of; manganese, turnover; rat, manganese absorption and excretion in; manganese concentration of tissues; turnover of tissue manganese in relation to dietary supply.

INTRODUCTION The remarkable constancy of tissue manganese (Mn) concentration over a wide range of dietary Mn supply reflects efficient homeostatic control (1,2). Radioisotope studies by Britton and Cotzias (3) led to the original and often cited hypothesis that Mn homeostasis would be regulated by a variable excretion via the digestive tract. Other reports (4-6), however, have emphasized that a variable efficiency of absorption is also an important factor in Mn homeostasis. On the basis of a factorial concept of trace element utilization and retention (7), it is evident that any change in the rate of true absorption of dietary Mn must be counterbalanced by an equivalent response in e n d o g e n o u s Mn excretion for maintaining stable concentrations at the tissue level. In this report, we present and discuss quantitative data on true absorption and e n d o g e n o u s fecal excretion of Mn over a wide range of dietary Mn supply in fast-growing rats. These parameters were determined following the approach of the radioisotope dilution procedure described in the preceding paper (8).

METHODS Treatments of Animals Forty-eight male weanling SPF Sprague-Dawley rats, purchased from a commercial source, with a mean initial live weight of 34.4 g, were first kept in groups of six animals per cage (metal-free). For 10 d they were fed a semipurified diet containing 1.5 ppm Mn on a dry basis. After 8 d, all animals were transferred to metal-free metabolism cages and kept individually until the end of the experiment. During this time they were fed twice daily (at 0730 and 1630). Mealtime was of sufficient length to aUow ad libitum consumption of the diet. Deionized water, supplemented with 0.14% NaCI to adjust osmolarity, was available at all times. The animal room was maintained at a constant temperature of 25~ and a relative humidity of about 60%. After the adaptation period of 2 d in the metabolism cages, eight rats each were assigned randomly to one of six different diets, containing by analysis 1.5, 4.5, 11.2, 35, 65, or 100 ppm Mn on a dry-matter basis. Three days after this dietary treatment had been started, each animal was inBiological Trace Element Research

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jected intramuscularly (left thigh) with a dose of 10 p,Ci carrier-free ~MnCI2, as previously described (8). Injection time was 1.5 h after the morning meal. A 16-d experimental period followed. Feces and urine were collected separately in 4-d periods: d 1-4, 5-8, 9-12, and 13-16. Dietary intake was assessed in corresponding 4-d periods, which, however, anteceded the fecal collection periods by 1 d to account for the approximate gastrointestinal transit time. At the end of the 16-d balance period, all rats were bled under ether anesthesia. Digestive tract, liver, pancreas, spleen, kidneys, testes, lungs, heart, left and right femur, and the skeletal muscle tissue surrounding the femora were isolated from each animal and weighed. The remaining body is referred to as "residual body" in this report. Serum was prepared from the blood. The blood clot was added to the residual body. All samples, including feces and urine, were stored at -20~ until analysis.

Diet Composition and preparation of the basal semipurified diet was the same as that described in the preceding paper (8): 25% casein, 28% sucrose, 28% maize starch, 4% cellulose, 7.7% coconut fat, 1% linoleic acid, 6.3% mineral and vitamin premixes, and DL-methionine. MnCI2-4H20 was used to establish dietary Mn concentrations above the basal level of 1.5 ppm.

Analyses Sample preparations and analytical procedures for assessing concentrations of stable Mn and radio-Mn were those described in the preceding paper (8). Urine samples and gastrointestinal tract were analyzed only with respect to 54Mn content. Skeletal muscle from the left thigh (site of 54Mn injection) and the right thigh of each animal did not differ in radioactivity. The same was true for the left and right femur. For analysis of stable Mn, lungs, heart, spleen, and testes were combined for each animal and are referred to as "pooled tissues." In the case of skeletal muscle, the samples from four animals each were pooled to facilitate Mn determination so that only two observations were available per treatment group.

Evaluation of Data Endogenous Mn excretion in feces and true absorption of dietary Mn were determined according to the radioisotopic procedure described in the preceding paper (8). Data were subjected to analysis of variance and simple regression analysis by common statistical procedures (9). Biological Trace Element Research

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RESULTS Mean live weight, growth rate, and food intake (Table 1) were not affected by dietary Mn supply during the 16-d experimental period. Tissue Mn turnover, however, was greatly influenced. This is evident from Fig. 1, showing regression lines of the specific activity of body Mn on dietary Mn concentration 16 d after the intramuscular ~ M n injection. This double-log graph illustrates that there were not only major differences among the various organs and tissues at each of the dietary Mn levels, but also that the specific activities markedly decreased with increasing Mn supply. Despite the straight-line relationships shown in Fig. 1, it must be realized that according to the distinctly exponential response, the decline of the specific activities of tissue Mn was, on a linear scale, most pronounced over the range of dietary Mn concentration covered by treatment groups I (1.5 ppm) to III (11.2 ppm). This is particularly true for the response of the Mn in liver and serum, whereas bone (femora) and skeletal muscle showed the least dynamics. This is also evident from the respective regression coefficients reported in the legend of Fig. 1. Bone and skeletal muscle were closely comparable with respect to the exponential rate of decrease of the specific activity, despite a 3.5-fold difference in the degree of labeling. Manganese of the pancreas, kidneys, and pooled tissues was, overall, highly labeled in comparison with the serum values and, except for group I, with the liver values. The observed mean values of the specific activity of serum and liver Mn in the six dietary treatment groups at the end of the 16-d experimental period are recorded in Table 2. These data (and also Fig. 1) indicate that the labeling of Mn in serum was markedly lower than that in liver of groups I-III. This relationship, however, reversed with the increase in dietary Mn concentration, so that the serum values of groups V and VI TABLE 1 Mean Live Weight, Growth Rate, and Food Intake of Rats During 16-d Experimental Period Group" Diet, ppm Mn

I

II

Ill

IV

V

VI

1.5

4.5

11.2

35

65

100

Live weight, g Initial, d 0 72.9 73.1 72.3 72.2 70.8 72.6 Final, d 16 173.3 174.1 172.3 172.9 172.4 1 7 5 . 2 Growth rate, g/d d 0-16 6.37 6.31 6.25 6.28 6.27 6.41 Food intake, g/d d 0-16 11.4 11.2 11.1 10.8 10.8 11.1

Overall mean

SEM, n = 8

72.3 173.4

_+1.97 ___3.34

6.30

_+0.13

11.1

+0.18

"No significant differences (p > 0.05) between group means in any of the parameters of this table. Biological Trace Element Research

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3oo 200 f

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2

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8 10

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15 20 Z,0 60 100 Dietory Mn concentration(ppr~

Fig. I. Regression of the specific activity of body Mn on dietary Mn concentration 16 d after intramuscular ~4Mn injection. The lines represent regression equations defined by In y = b,, + lh In x, where ,t/ represents the specific activity and x dietary Mn concentration. Intercept, regression, and correlation coefficients (b,,/q, r, respectively) are in the case of skeletal muscle 4.60, - 0.26, -0.86; pancreas, 5.58, -0.52, -0.93, kidneys, 5.70, - 0.59, -0.95; residual body (i.e., whole body without viscera and testes), 4.09, -0.37, -0.91; femora, 3.35, - 0.25, - 0.91; serum 5.13, - 0.75, - 0.98; and liver, 5.84, - 0.98, - 0.98. w e r e significantly h i g h e r t h a n the respective liver values. It is further evid e n t from Table 2 that fecal M n excreted from d 9 to 16 after 54Mn a d m i n istration w a s labeled to a very m u c h lower extent t h a n s e r u m or liver Mn at the e n d of d 16. Accordingly, m e a n p e r c e n t a g e s for e n d o g e n o u s fecal M n s h o w n in this table are, overall, fairly low. O n the basis that the labeling of s e r u m M n of d 16 is r e p r e s e n t a t i v e for the e n d o g e n o u s M n in the feces of d 9-16, a m e a n of 6.3% of the total fecal M n was of e n d o g e n o u s origin in the case of g r o u p I a n d 1.5% in the case of g r o u p VI. This decrease w i t h increasing dietary M n s u p p l y is a p p r o x i m a t e l y linear (r = - 0 . 8 2 ; p < 0.001). W h e n the specific activity of liver M n is u s e d instead of the s e r u m values for calculating the c o n t r i b u t i o n of e n d o g e n o u s sources to the fecal excretion of the metal, the resulting m e a n percentages s h o w a s o m e w h a t smaller r a n g e of variation a m o n g t r e a t m e n t g r o u p s (Table 2), a n d their inverse relationship with dietary M n s u p p l y is less p r o n o u n c e d (r = - 0 . 4 8 ; p < 0.01). In Table 3, results on intake, e n d o g e n o u s fecal excretion, a n d abs o r p t i o n of M n are p r e s e n t e d for the 8-d balance p e r i o d f r o m d 9 to 16. T h e increase in m e a n daily M n intake from g r o u p I to VI is very closely Biological Trace Element Research

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TABLE 2 Mean Specific Activity of Mn in Feces, Serum, and Liver and Percent Fecal Mn of Endogenous Origin Computed on Basis of Specific Activity of Serum and Liver Mn 16 d After r~Mn Injection Group Diet, ppm Mn

1

I1

III

IV

V

VI

1.5

4.5

11.2

35

65

100

Specific activity, cpm ~Mn/ng Mn Feces, d 9-16 8.4"' SEM _+0.5 Serum, d 16 Liver, d 16 SEM: Difference:, p

135.ff' 225.7'' _+9.9 0.10

7.80a'v 5.72 'r-'~ -+0.58