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Essential Trace Metals and Specific Organ Weights in Diet-Restricted and Ad Lib-Fed Rats BRIAN C. LEE, KLAU$ L. STEMMER*, AND HAROLD G. PETERING

Department of Environmental Health, University of Cincinnati College of Medicine, 3223 Eden Ave., Cincinnati, OH 45267-0056 Received January 30, 1984; Accepted January 29, 1985

ABSTRACT Manganese, copper, iron, zinc, and specific organ weights (relative to body weight) of the heart, liver, and kidney were compared between a 65% diet-restricted group and an ad lib-fed control group of young male Sprague-Dawley rats. Elevated concentrations of Cu (12%) in the liver, and of Mn (12-26%) and Fe (17-69%) in all three organs, occurred in the diet-restricted group. Specific heart weight was unchanged despite the 40% difference in group body weights. Specific liver weight decreased 13% and specific kidney weight increased 13%. I n d e x Entries: Manganese, in heart, liver, and kidney; trace metals in heart, liver, kidney; restricted food intake; diet restriction; heart, liver, kidney specific weight or relative weight; copper, in heart, liver, and kidney; iron, in heart, liver, and kidney; zinc, in heart, liver, and kidney; heart, Mn, Cu, Zn, and Fe in; liver, Mn, Cu, Zn, and Fe in; kidney, Mn, Cu, Zn, and Fe in.

INTRODUCTION Reduced food intake, despite ad libitum access, is an occasional response in experimental animals during toxicologic studies. When the in*Authorto whom

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take is significantly reduced, the investigator m u s t distinguish w h e t h e r the results obtained were caused by the toxicant, diminished food intake, or both. Monitoring of food consumption then becomes essential. It has been generally assumed that a pair-feeding design cancels the effects of diet c o n s u m p t i o n between groups. But an altered metabolism can occur in the pair-fed control group, which could alter experimental results. A diet-restriction experiment was conducted to examine the effects of decreased food intake on essential trace metals and specific organ weights. Vital organs are k n o w n to be sensitive to alterations in trace metal metabolism. Since the source of essential trace metals is primarily dietary, it was reasonable to seek changes in the meta{s during diet restriction. Manganese, copper, iron, and zinc were selected because of their essentiality and their toxicological relevance in oxidative d a m a g e and defense mechanisms. Organ weights are used routinely as initial evidence of a toxic response. Effects on certain organ weights can be better described as a proportion of the total body weight. "Specific organ weight" assumes the relationship b e t w e e n an organ and body weight remains relatively constant in a normal animal beyond a critical stage in life, generally weaning. Changes in specific organ weights occur as the result of various treatments. Specific heart weight (SHW) increases with cardiotoxicants, e.g., c a d m i u m (1), during suboptimal nutrient intake, e.g., dietary copper deficiency (2), and u n d e r environmental changes, e.g., high altitude (3). Specific liver weight (SLW) is higher after exposure to microsomal enz y m e oxidizing system inducers, such as pesticides (4) or anesthetic and tranquilizing drugs (5). Specific kidney weight (SKW) has been s h o w n to increase with dietary lithium exposure (6) and copper deficiency (7).

METHODS Animals and Diet Male Sprague-Dawley weanling rats (Charles River CD, avg wt = 80 g) w e r e fed Purina Lab Rodent C h o w 5001, 1/,_-in. checkers, ad lib for 8 d. The c h o w was found to contain (in txg/g diet): Mn = 78.8, Cu = 16.4, Fe = 572, Zn = 77.5. Drinking water was available ad lib. Rats w e r e h o u s e d individually in stainless steel cages with s u s p e n d e d mesh floors. The fluorescent lighting cycle was 12L:12D. On the ninth day, the rats (avg wt = 140 g) were r a n d o m l y assigned to a diet-restricted or an ad lib-fed group. Food consumption of the dietrestricted group was limited to approximately 35% of the c o n s u m p t i o n of the ad lib group. After the diet-restricted group had lost 20% of their prerestriction body weight, all animals were sacrificed on the 16-17th day.

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Organ Weights Excised hearts w e r e cleared of blood in their chambers with 0.9% NaC1, a n d t h e n o p e n e d to r e m o v e any a d h e r e n t blood. The heart, liver, a n d the right k i d n e y were blotted on paper toweling and w e i g h e d .

Trace Metal Analysis Glassware was soaked o v e r n i g h t in 10~ nitric acid and rinsed in deionized water to r e m o v e trace metals. The heart, right kidney, a n d a section of the frontal lobe of the liver from each rat were desiccated to constant w e i g h t in an electric oven at 110~ (approximately 3 d for heart a n d kidney, 5 d for liver). Diet samples were pulverized in a plastic bag. All samples were digested in an initial 70 to final 110~ refluxing mixture of nine v o l u m e s concentrated nitric acid and one v o l u m e concentrated perchloric acid over a period of 3-5 d. The digests were e v a p o r a t e d to near d r y n e s s a n d rediluted in 10% nitric acid. Recoveries were c h e c k e d with National Bureau of Standards Bovine Liver Reference Material 1577. The metals were analyzed on a Perkin-Elmer 403 air/acetylene flame atomic absorption s p e c t r o p h o t o m e t e r . Standard curves were c o m p u t e d by linear regression of dilutions of atomic absorption 1000 ~g/mL Fisher Scientific certified reference solutions.

RESULTS The concentration of trace metals in the organs are s h o w n in Table 1 a n d are r e p r e s e n t e d as a percentage of the ad lib control values in Fig. 1. Mn concentrations were increased 26% in the heart a n d liver, b u t only 12% in the kidney. Cu concentrations were significantly c h a n g e d only in the liver, which experienced a 12% increase. Fe concentrations were elevated to various degrees: 17% in the heart, 44% in the kidney, 69% in the liver. Zn concentrations, however, r e m a i n e d stable in all three organs. The total a m o u n t of trace metals in each organ is s h o w n in Table 2 a n d r e p r e s e n t e d as a percentage of the ad lib control values in Fig. 2. The total a m o u n t s decreased in all organs, except for Fe. The values for Cu in the liver s h o u l d be considered an estimate because of the u n e q u a l distrib u t i o n b e t w e e n the lobes (8). Iron was c o n s e r v e d in liver and k i d n e y , b u t not in heart. Heart retained the least a m o u n t of Fe as a percentage of the ad lib control value. The livers of the diet-restricted animals retained lower p e c e n t a g e s of Mn, Cu, and Zn. A n i m a l a n d sample n u m b e r s were rechecked to confirm p r o p e r identification. The diet-restricted group exhibited reductions of the weights of all three o r g a n s (Table 3). Relative to b o d y weight, each organ r e s p o n d e d

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TABLE 1 Concentration of Essential Trace Metals as Determined by Atomic Absorption Spectrophotometry in p.g/g Dry. Tissue" Tissue Heart Restricted Ad lib Liver Restricted Ad lib Kidney Restricted Ad lib

Mn

Cu

Fe

Zn

2.47* (0.35) 1.96 (0.09)

23.61 (1.12) 21.66 (2.25)

298,* (26.) 254. (23.)

95.8 (3.3) 88.5 (11.3)

10.11"* (0.83) 7.96 (0.87)

16.21" (1.10) 14.51 (1.05)

562.* (188.) 332. (53.)

108.7 (10.4) 102.5 (8.7)

4.46* (0.24) 4.00 (0.30)

19.86 (1.95) 22.30 (3.85)

310.* (54.) 216. (40.)

75.2 (4,1) 83.3 (9.7)

~(Standard deviations) are underneath mean values. Student's '~ t-test probabilities: *p < .05 **p < .01 ***p

Essential trace metals and specific organ weights in diet-restricted and ad lib-fed rats.

Manganese, copper, iron, zinc, and specific organ weights (relative to body weight) of the heart, liver, and kidney were compared between a 65% diet-r...
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