copper absorption, endogenous excretion, and distribution in sprague-dawley and lean (fa/fa) zucker...

~)Copyright 1996 by Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/96/5301-3--0261 $10.00

Copper Absorption, Endogenous Excretion, and Distribution

in Sprague-Dawley and Lean (Fa/Fa) Zucker Rats

GARY D. MILLER, CARL L. KEEN, JUDITH S. STERN, AND JANET Y. URIU-HARE*

Department of Nutrition~Meyer Hall, University of California, Davis, Davis, CA 95616

Received June 9, 1995; Accepted July 30, 1995

ABSTRACT

We previously observed a rapid reduction in plasma ceruloplas- rain activity in lean Zucker (Fa/Fa) rats fed a marginal copper (Cu)- deficient diet compared to similarly fed obese Zucker (fa/fa) and lean Sprague-Dawley rats. In an effort to understand the mechanisms underlying this response, we utilized the isotope dilution method to investigate the absorption and excretion of Cu in lean Zucker rats fed control and marginal Cu diets. Sprague-Dawley (SD) and homozygous lean Zucker rats were fed either a Cu-adequate (Cont; 7.5 ~tg Cu/g diet) or a low Cu (Low; 1.1 ~tg Cu/g diet) casein-based diet for 23 d. Two weeks following initiation of the dietary treatment, each rat was injected intramuscularly (im) with 11.2 ~tCi of 67Cu. Urine and feces were collected daily. On the 9th d following isotope injection, rats were killed and tissues collected. Significant dietary effects were observed in the relative absorption and endogenous fecal excretion of 67Cu. The tissue distributions of nonisotopic Cu and 67Cu activity were also different between dietary treatments. Tissues from rats fed the low-Cu diet typically had high concentrations of 67Cu and low concentrations of nonisotopic Cu compared to controls. An increase in relative 67Cu absorption was evident for rats fed the low-Cu diet (57.2 and 39.3%, for SD Low, Zucker Low, respectively, and 17.9, and 28.5% SD Cont and Zucker Cont, respectively). Rats fed the low-Cu diet also had reductions in endogenous fecal excretion of 67Cu compared to their respective controls. Although strain effects were not

*Author to whom all correspondence and reprint requests should be addressed.

Biological Trace Element Research 261 Vol. 53, 1996

262 Miller et al.

evident for either percent Cu absorption or endogenous fecal Cu excretion, the relative adaptive changes appeared more marked for the Sprague-Dawley rats compared to the lean Zucker rats.

Index Entries: Copper; absorption; excretion; Zucker; rats.

INTRODUCTION

In a recent study of copper (Cu) metabolism in Zucker rats, we observed a marked difference in the handling of Cu between the lean (Fa/Fa) and obese (fa/fa) phenotypes when rats were fed a marginal Cu diet (3 ~tg Cu/g diet) for 2 mo (1). At the end of the experiment, plasma and liver Cu concentrations, and plasma ceruloplasmin (Cp) activity, were significantly lower in lean Zucker rats compared to the obese rats (97, 85, and 55% less, respectively). The lower Cu status of the lean Zucker rat was also reflected by a 67% decrease in liver CuZn superoxide dismutase (SOD) activity compared to obese Zuckers. To explore further the variant response for Cu in the lean Zucker rat, Keen and coworkers fed Sprague-Dawley (SD) and lean Zucker rats either a stock diet or the marginal Cu diet (3 ~tg Cu /g diet) for 2 wk. Cp activity was similar for SD rats fed either the stock or marginal Cu diet, whereas Cp activity was depressed in Zucker rats fed the marginal Cu diet compared to the stock diet. Liver Cu concentrations and liver CuZn SOD activity were also reduced in Zucker rats fed the marginal Cu diet compared to their stock fed controls, suggesting a compromise in functional capacity. No such dietary relationship was observed for SD rats.

Consistent with these findings, homo- and heterozygote lean Zucker (Fa/Fa and Fa/fa, respectively) rats fed a diet containing 1.1 ~tg Cu /g diet showed a marked decrease in Cp activity from baseline measurements beginning as early as 2 d (32% reduction) following initiation of the diet (Miller, unpublished data). By day 4, an 80-84% reduction in Cp activity was observed, and it was further reduced to 94-98% of baseline at day 7. In contrast to the lean genotypes, no differences in plasma Cp activity was observed for obese Zucker rats pair-fed for up to 35 d with a diet containing 1.1 ~g Cu /g diet. Although Zucker rats have been stud- ied in detail with regard to their obesity, little research has been done examining their trace mineral metabolism, particularly for Cu.

Studying the different responses to marginal Cu diets in various rat strains can be insightful for investigating Cu metabolism. Different strains and even different sexes in the same strain metabolize Cu differ- ently (2-8). The current study investigates Cu metabolism in the lean Zucker and SD rat, specifically the absorption, excretion, and tissue distribution of Cu, in an effort to explain differences in the handling of a low-Cu diet among the two strains. It is hypothesized that the differences in Cu homeostasis for Zucker rats is due, in part, to an alteration in either the absorption and/or endogenous excretion of this trace mineral.

Biological Trace Element Research Vol. 53, 1996

Cu Absorption in Rats 263

Demonstration of differences in the biliary excretion of Cu among different rat strains lends support for this hypothesis (3,5-7).

Cu absorption and endogenous excretion in this investigation were determined by the isotope dilution method. This method allows the simultaneous calculation of both absorption and endogenous fecal excretion of Cu by discriminating between unabsorbed dietary Cu in feces from fecal Cu of endogenous origin. This procedure is widely used in the study of zinc (Zn) metabolism (9), and has recently been adapted and validated for use with Cu (10).

METHODS

Three to 4-wk-old male rats from the Sprague-Dawley strain (Charles River, Wilmington, MA) and the homozygous lean (Fa/Fa) Zucker strain (NIH-Clinical Nutrition Research Unit, Animal Models Core Lab, University of California, Davis) were used to investigate Cu absorption, excretion, and tissue distribution under conditions of adequate and low-Cu diets. On arrival, rats were housed in hanging stainless-steel wire cages in a temperature (22 _+ 2~ and light-controlled room (12 h light/dark cycle; lights on at 0700 h) and were fed a stock diet (Formulab 5008, Labdiet PMI Feed, Inc., St. Louis, MO) for 2 d to adapt to laboratory conditions. All rats were fed a Cu-adequate (7.5 ~tg Cu/g diet) diet for 4 d to establish a baseline period. Following this baseline period, rats from each strain were randomly assigned to one of two diet groups: the Cu-adequate casein-based diet (Cont) or a low-Cu (1.1 ~tg Cu/g diet) casein-based diet (Low) (Table 1). Diets differed only with respect to their Cu content. Rats were divided into four groups (n = 4) designated by their strain and dietary treatment: SD Cont, SD Low, Zucker Cont, and Zucker Low. Throughout the experiment, all animals had unrestricted access to deionized water and diet. Food intake was measured daily.

Two weeks following the initiation of the two dietary treatments, each rat was injected im in the hindleg with 11.2 ~tCi of 67Cu (SA = 0.5 ~tCi/~tg) in 0.9% saline-5 mM glycine (University Missouri Reactor Cen- ter, Columbia, MO). Animals were then placed in metabolic cages for daily urine and feces collection. The amount of radioactivity in fecal and urine samples was determined daily for the following 9 d (Minaxi Auto- Gamma 5000 Series Gamma Counter, Packard Instrument Co. Downers Grove, IL). On the 9th d following isotope injection, animals were asphyx- iated with CO2 and killed by cardiac puncture. Blood was collected in heparinized syringes, centrifuged (4~ for 20 min at 1700g), and aliquots of plasma and red blood cells (RBC) were placed in scintillation vials for measurement of 67Cu activity. The liver and heart were perfused with 0.9% NaC1 and then removed. Other tissues excised included kidney, tibia, brain, gastrocnemius, spleen, and a small piece of shaven skin from


264 Miller et al.

Table 1 Composition of the 7.5 ~tg Cu/g Diet

Diet Composition (g/ka diefl

Casein 21.0

DL-Methionine 0.3

Corn starch 20.0

Glucose 39.2

Corn oil 8.0

Mineral mix* 6.0

Cellulose 4.0

Vitamin mixt 1.5

*Mineral mixture for diet contained (g/kg mix): CaCO3, 238.90; MgSO4, 97.0; CaHPO4, 60.0; K2HPO4, 321.0; FeSO4 �9 7H20, 10.0; NaC1, 43.0; CuSO4 �9 5H20, 0.66; CrK(SO4)2 �9 12H20, 0.40; MnSO4 - H20, 2.30; KI, 0.20; ZnCO3, 1.60; Na2SeO3, 0.0018; Glucose, 224.94.

tVitamin mixture contained (g/kg mix): inositol, 25.0; ascorbic acid, 5.0; calcium pantothenate, 2.5; thi- amine hydrochloride, 1.5; pyridoxine hydrochloride, 1.5; nicotinic acid, 1.5; menadione, 1.25, riboflavin, 0.5; p-aminobenzoic acid, 0.5; folic acid, 0.03; biotin, 0.0125; Rovimix E-50%, 7.8; Rovimix A-650, 0.205; Rovimix AD3 A650/D325, 0.205; Merck B12+ mannitol, 1.50; choline chloride (70%; mL/kg), 71.50 (Sources: Rovimix, Hoffman-LaRoche, Nutley, NJ; Merck & Co., Rahway, NJ); glucose, 879.49.

the abdominal region. All tissues were placed in scintillation vials for determination of 67Cu activity. Aliquots of these tissues were then used for analysis of Cu, iron (Fe), manganese (Mn), and Zn concentrations. For trace mineral analysis, tissues were wet ashed (11), and their mineral concentrations were determined by flame atomic absorption spectrophotom- etry (Model 551, Thermo-Jarrel Ash, Wilmington, MA).

Owen (12) has reported the relative contribution of bone (14.2%), muscle (39.0%), plasma (3.4%), RBC (2.2%), and skin (16.2%) to total body wt. These percentages were used to calculate the total pool for these tissues.

Absorption of Cu was calculated using the formula:

I - F + (F- Sf/Sm)/I (1)

where I = mean daily intake of Cu for days 7, 8, and 9; F = mean fecal Cu excretion for days 7, 8, and 9; Sf = mean SA of feces for days 7, 8, and 9; and Sm = the combined mean SA of the sampled tissues (bone, brain,

Biological Trace Element Research VoL 53, 1996


heart, kidney, liver, muscle, and spleen). Using the combined mean for specific activity from these seven tissues is a minor variance to the pro- cedures previously used. Both Johnson and Lee (10) and Johnson (13) measured the SA for liver and kidney and used these two tissues for determining Sm. Use of these two tissues was based on observations that the combined mean SA for kidney and liver was similar to the combined mean SA of other tissues. In our study, the combined mean SA for liver and kidney was not representative of the combined SA of the remaining sampled tissues. Thus, we determined the combined mean SA of all sampled tissues in calculating absorption. Endogenous excretion is expressed by the formula in parentheses: F �9 Sf/Sm.

A two-factor ANOVA was used to evaluate the influence of dietary Cu and rat strain on most dependent variables. Data were also analyzed by one-factor ANOVA. When the F value was significant (p < 0.05), group differences of means were determined by post hoc comparisons (Student Newman Keuls). All analyses were performed on SAS, Inc. software (Cary, NC).

RESULTS

Body and Tissue Weights

Although all rats were similar in age, riD rats were significantly heavier than Zucker rats throughout the experiment (Table 2). Tissue weights for heart, kidney, and liver were higher for SDs compared to Zuckers. However, the relative mass of the tissues (tissue wt: body wt) was similar between the strains. The level of Cu in the diet did not alter body or tissue wt.

Food and Cu Intake

Daily food intake for the 9 d following the isotope injection was similar for the two dietary treatments (Fig. 1). However, by two-way ANOVA, there was a significant strain effect. Overall, SD rats, which were heavier than Zucker rats, ate significantly more than Zucker rats on days 1, 4, 5, 8, and 9. The equation used in determining Cu absorption requires the mean dietary intake of Cu for the 3 d prior to killing the animals. The values used in the calculations were: 172.7 _+ 14.1 (SD Cont), 23.2 + 0.8 (SD Low), 153.1 +_ 11.7 (Zucker Cont), and 19.5 _+ 1.0 ~tg Cu (Zucker Low), which differed only with respect to dietary treatment.

Cu Absorption and Endogenous Excretion

By two-way ANOVA, relative Cu absorption (Fig. 2A) was higher for the two groups receiving the low-Cu diet (57.2 and 39.3% for SD Low and Zucker Low, respectively) compared to groups fed an adequate-Cu


266 Miller et al.

Table 2 Effect of Dietary Cu on Body and Tissue Wts in Sprague-Dawley

and Lean Zucker Rats (Fa/Fa)

Tissues SD Cont

Initial Body

Weight (g) 227.0 _+ 5.6 a

Groups

SD Low Zucker Cont Zucker Low

231.3-+ 6.1 a 189.8-+ 11.1 b 192.8+ 7.6 b

Final Body

Weight (g) 363.0-+ 13.5 a

Heart (g) 1.33 + 0.09 a

Kidney (g) 2.97 + 0.13 a

Liver (g) 14.83 + 0.51 a

Spleen (g) 0.93 + 0.11

Heart:Body Wt (%) 0.37 + 0.02

Kidney:Body Wt(% )0.82 -+ 0.02

Liver:Body Wt 1%) 4.09 -+ 0.09

Spleen:Body Wt(%/ 0.25 -+ 0.02

369.3+ 24.4 a 276.0+ 9.2 b 274.8+ 12.2 b

1.35 + 0.16 a 1.02 + 0.05 b 1.05 + 0.05 b

3.18+ 0.23 a 2.26+ 0.02 b 2.26_+ 0.09 b

19.93 _+ 1.73 a 10.75 _+ 0.70 b 10.65 _+ 0.88 b

1.08 + 0.21 0.54 _+ 0.02 0.93 _+ 0.35

0.36 _+ 0.02 0.37 + 0.01 0.38 _+ 0.02

0.86 + 0.03 0.82 -+ 0.02 0.83 -+ 0.04

4.28 + 0.20 3.88 + 0.15 3.86 -+ 0.20

0.30 -+ 0.06 0.20 -+ 0.00 0.34 -+ 0.14

Results are means _+ SEM. Means with different superscripts within a row are significantly different

(p _< 0.05) from each other. Rats were fed a diet adequate in Cu (Cont) or low in Cu (Low).

diet (17.9 and 28.5% for SD Cont and Zucker Cont, respectively). Percent absorpt ion did not differ for the two strains. However , the s train-by-diet interact ion approached statistical significance at p = 0.074. Post hoc compar isons revealed that Cu absorpt ion values for the SD Low rats was sig- nif icantly h igher than for the SD Cont and Zucker Cont rats, bu t the increase in Cu absorpt ion for the Zucker Low group was in te rmedia te and did not differ statistically f rom either the SD Low or the Cu adequa te groups. Post hoc compar isons of net Cu absorpt ion (SD Cont, 29.2 + 6.9; SD Low, 13.1 + 1.1; Zucker Cont, 45.4 _+ 11.9; and Zucker Low, 7.8 + 2.1 ~tg/d) s h o w e d that the SD Low and Zucker Low groups were lowest, the Zucker Cont group was highest , and the SD Cont g roup was in termedi- ate and not different f rom the other three groups. The average endogenous fecal excret ion of Cu was significantly lower in the SD Low and Zucker Low groups compared to the SD Cont and Zucker Cont groups



30"

20 t -

r 15

�9 lO 0

SD Cont

�9 SD Low

----t:2--- Zucker Cont

-- Zucker Low

i i t i L i , i l

! 2 3 4 5 6 7 8 9

D a y s

Fig. 1. Effect of rat strain and dietary copper (Cu) concentrations on daily food intake across the 9-d period following isotope injection. Sprague-Dawley (SD) and Zucker lean rats (Fa/Fa) were fed a diet adequate in Cu (Cont) or low in Cu (Low). Sprague-Dawley rats ate significantly more food than Zucker rats on days 1, 4, 5, 8, and 9.

(Fig. 2B). No strain differences were apparent for net Cu absorption or excretion following two-way ANOVA.

67Cu Excretion

Fecal and urine samples constituted a 24-h collection period with the exception of day 1, which spanned a 12-h period. Day 1 measurements were thus omitted from the analysis and figure presentations.

Fecal

Daily fecal excretion of 6 7 C u w a s lower for the SD Low and Zucker Low groups compared to their respective controls for days 2--4, and days 6-7 following the isotope injection (Fig. 3A). No strain effect was observed. Dietary treatment did not alter fecal 67Cu excretion when expressed per gram of feces (data not shown).

Urine Daily urinary 6 7 C u activities for the riD Low and Zucker Low groups

were significantly lower than for the SD Cont and Zucker Cont groups on days 3, 4, and 5 following the isotope injection (Fig. 3B). No differences were observed among the two strains of rats at any time-point.


268 Miller et al.

~d

C

L .

m r

70

60

50

40

30'

20'

10

A

b

T

SD Cont SD Low ZU Cont

ab

T

ZU Low

L.

OdD

0

r t_

0

C

N

60" [3

5 0

40'

30'

20'

10

SD Cont SD Low ZU Coat ZU Low

Fig. 2. Effect of rat strain and dietary Cu concentrations on relative Cu absorption {A) and endogenous Cu excretion (13). Sprague-Dawley (SD) and Zucker lean rats (Fa/Fa) were fed a diet adequate in Cu (Cont) or low in Cu (Low). Values with different letters are significantly different from each other.

Fecal Copper Concentration

Nonisotopic fecal Cu excretion was significantly lower over all 9 d of collection in the 2 low-Cu groups compared to the Cu-adequate controls (Fig. 4). Post hoc comparison analysis revealed that on day 8, daily



70"

60"

50"

.~ 40"

= 30' t ' , -

2 0

I0 �84

0

20"

b . ;

A -'-'O--- SD Cont

�9 SD Low

Zucker Cont

-- Zucker Low

/ i * l , , , i

2 3 4 5 6 7 8 9

B /

2 3

SD Cont

�9 SD Low

-----D--- Zucker Cont

-- Zucker Low

4 5 6 7 8 9

Days

Fig. 3. Fecal (A) and urinary (B) 67Cu activity for days 2-9 following isotope injection. Sprague-Dawley (SD) and Zucker lean rats (Fa/Fa) were fed a diet adequate in Cu (Cont) or low in Cu (Low).

Cu excretion was higher for the SD Cont group than for the other three groups. There were no differences in fecal Cu excretion between the Zucker Low group and the SD Low groups.

Tissue and Fecal Specific Activity

Rats fed the low-Cu diet had significantly higher tissue specific activity (nCi 67Cu/~tg Cu) than Cu-adequate animals in all tissues analyzed by two-way ANOVA, with the exception of the heart (Fig. 5A and


270 Miller et al.

.o

t .

=

c~

200"

100"

0 l / , , 2 3

" - - < > ' - SD Corn

�9 SD Low

Zucker Cont

�9 Zucker Low

i J

4 5 6 7 8

1

J

9

Fig. 4. Effect of rat strain and dietary Cu concentrations on fecal Cu excretion, ~tg Cu/d. Sprague-Dawley (SD) and Zucker lean rats (Fa/Fa) were fed a diet adequate in Cu (Cont) or low in Cu (Low).

B). Further analysis by post hoc comparisons revealed that for all tissues (excluding the heart), the specific activity was significantly higher in the Zucker Low group than in the two control groups. Tissue specific activities in SD rats fed the low-Cu diet were intermediate and not different from the other three groups for bone, muscle, plasma, liver, and RBCs. The SD Low group was significantly higher than the two adequate Cu groups for brain and kidney; t-test comparisons demonstrated significantly higher specific activity in the SD Low group than the SD Cont group for bone, brain, muscle, plasma, kidney, RBCs, and spleen, although liver approached significance (p = 0.067). Strain differences were not apparent for any tissue by two-way ANOVA.

Specific activity in feces was determined for the final 9 d of the experiment. The average value for days 7, 8, and 9 was utilized in calculating Cu absorption and endogenous excretion (Fig. 6). Analysis by two-way ANOVA revealed no strain differences, but diet effects were evident from days 3-9. Specific activity was higher for the low-Cu groups compared to their respective controls.

67Cu Tissue Distribution

The tissue distribution of the 67Cu isotope was affected by the level of Cu in the diet (Table 3). In groups fed the low-Cu diet, most tissues had an increased 67Cu activity on a relative basis (nCi/g tissue) compared to controls (bone, brain, kidney, muscle, skin, and spleen). Values tended to be higher in liver and RBCs, but this did not reach statistical



4"

~sts

t~

2" .,t

.r r

e~

O2

16-

14" M .~ �9

r, ,-

~, 1 2 -

lo ' ~ .

~

K

IA

[] SD Cont

Irl SD Low

�9 Zucker Cont

[] Zucker Low

a & a

Bone Brain Heart

B

[] SD Cont

[] SD Low

[] Zucker Cont

[] Zucker Low

& a a

T ab

ab

Kidney Liver RBC

ab

Muscle Plasma

Tissues

b b

Spleen

Fig. 5. (A and B) Tissue specific activity (nCi 67Cu/~tg Cu). Sprague- Dawley (SD) and Zucker lean rats (Fa/Fa) were fed a diet adequate in Cu (Cont) or low in Cu (Low). Values with different letters within each tissue are significantly different from each other.

significance. In contrast, plasma 67Cu activity was significantly decreased in the low-Cu groups compared to their respective control groups. No strain differences were observed for these data.

Total body activity of 67Cu was determined by adding the activity for the calculated pool sizes (when appropriate) and all whole tissues collected. Percent distribution of 67Cu was calculated for each tissue by dividing the activity for the respective tissue by the total 67Cu activity.


272 Miller et al.

..=

o ~

~

~

r~

e~

1.4-

1.2 �84

1.0

0.8 t

0.61

0.4"

0 * 2 '

0.0

SD Cont

�9 SD Low ---D--- Zucker Cont

-- Zucker Low

/ t : , , t t t ,

2 3 4 5 6 7 8 9

Days

Fig. 6. Specific activity in feces (nCi 67Cu/~tg Cu) for days 2-9 following isotope injection. Sprague-Dawley (SD) and Zucker lean rats (Fa/Fa) were fed a diet adequate in Cu (Cont) or low in Cu (Low).

Two-way ANOVA revealed significant diet effects for several tissues, but strain effects were not demonstrated. Brain and skin from rats fed the low-Cu diet retained a higher percentage of the 67Cu dose than did these tissues from rats fed the adequate-Cu diet (Table 3). The percent distribution for plasma was significantly lower for the SD Low and Zucker Low groups than for their respective controls. The percent distribution of the 67Cu activity in heart was highest for SD Cont, with no differences evident among the other groups. No differences in other tissues collected were evident.

Tissue Trace Minerals

Copper By two-way ANOVA, rats in the low-Cu groups had lower Cu con-

centrations (nmol Cu /g tissue) in all tissues (except bone and brain) compared to the groups fed the Cu-adequate diet (Table 4). In the heart, post hoc comparisons showed that the Zucker Low group had significantly lower Cu concentrations than the SD Cont group. The SD Low and Zucker Cont groups were intermediate and did not differ significantly from the other two groups�9 In other tissues that demonstrated a significant diet effect, post hoc comparisons did not reveal further differences among groups.


Cu A b s o r p t i o n in Ra t s 2 7 3

Table 3 Effect of Dietary Cu on Distr ibution of 67Cu in Sprague-Dawley

and Lean Zucker Rats (Fa/Fa) Expressed as n C i / g Tissue or % Distr ibution

nCi/gram tissue Groups

Tissues SD Cont $D Low Zucker Cont

Bone 0.55• 0.12 b 0.85+ 0.12 a 0.61+ 0.12 b

Brain 0.29• 0.03 b 0.59-+ 0.12 a 0.27_+ 0.03 b

Heart 1.84+ 0.22 1.44-+ 0.20 1.66+ 0.17

Kidney 6.00_+ 1.57 b 9.40_+ 2.31 a 6.78-i- 1.21 b

Liver 2.98+ 0.76 4.18-+ 0.34 3.86+ 0.70

Muscle 0.57+ 0.08 b 0.72• 0.08 a 0.57• 0.07 b

Plasma 0.83+ 0.23 a 0.39_+ 0.22 b 0.94+ 0.23 a

RBC 0.53• 0.I0 0.62+ 0.14 0.54_+ 0.06

Skin 0.47+ 0.10 b

Spleen 1.19-t- 0.27 b

0.76+ 0.14 ab 0.47+ 0.I0 b

1.62_+ 0.17 ab 1.54• 0.15 b

A_NOVA, p-value

Diet (D)

Strain (S)

Zucker Low D X $

0.99+ 0.16 a 0.0253

NS

NS

0.61__. 0.02 a 0.0004

NS

NS

1.32_+ 0.15 NS

NS

NS

12.12+ 0.58 a 0.0155

N$

NS

5.66_+ 1.01 NS

NS

NS

0.80+ 0.07 a 0.0234

NS

NS

0.24_+ 0.05 b 0.0141

NS

NS

0.77_+ 0.29 NS

NS

NS

1.00_+ 0.09 a 0.0025

NS

NS

2.29• 0.35 a 0.0355

NS

NS

Results are means _+ SEM. Means with different superscripts within a row are significantly different (p < 0.05)

from each other. Rats were fed a diet adequate in Cu (Cont) or low in Cu (Low).


2 7 4 Miller e t al.

Table 3 (continued)

ANOVA, p-value

Diet (D)

Percent Distribution of 67Cu Groups Strain (S)

Tissues SD Cont SD Low Zucker Cont Zucker Low D X S

Bone 13.34+ 0.37 14.87+ 0.79 13.92+ 1.37 14.64+ 1.46 NS

NS

NS

Brain 0.25+ 0.01 b 0.37+ 0.06 a 0.25+ 0.04 b 0.39+ 0.03 a 0.0085

NS

NS

Heart 1.19+ 0.17 a 0.65+ 0.13 bc 0.98+ 0.07 bc 0.51+ 0.04 c 0.0009

NS

NS

Kidney 7.81+ 1.04 9.55+ 1.82 8.59+ 0.95 10.27+ 0.34 NS

NS

NS

Liver 19.39+ 1.66 22.00+ 2.35 22.86+ 0.23 21.76+ 1.68 NS

NS

NS

Muscle 38.50+ 3.42 34.23+ 2.89 34.69+ 2.17 31.76+ 0.72 NS

NS

NS

Plasma 4.37+ 0.48 a 1.66+ 0.92 b 4.71+ 0.54 a 0.79+ 0.15 b 0.0001

NS

NS

RBC 2.03+ 0.15 1.70+ 0.40 1.92+ 0.11 1.70+ 0.55 NS

NS

NS

Skin 12.62+ 0.52 b 14.38+ 1.61 a I i .60+ 0.77 b 17.31+ 2.73 a 0.0429

NS

NS

Spleen 0.49+ 0.03 0.58+ 0.i1 0.48+ 0.02 0.85+ 0.41 NS

NS

NS

Results are means + SEM. Means with different superscripts within a row are significantly different (p < 0.05) from

each other. Rats were fed a diet adequate in Cu (Cont) or low in Cu (Low).



Iron, Manganese, and Zinc Dietary Cu intake did not statistically influence Fe, Mn, or Zn con-

centrations in any of the tissues analyzed (data not shown).

DISCUSSION

The concentration of Cu in the diet altered Cu metabolism in both rat strains. Twenty-three days on a low-Cu diet compromised Cu status as assessed by low Cu concentrations in plasma and liver. Evidence of adaptive changes for rats fed the low-Cu diet was shown by an increase in their percentage of Cu absorption, and a decrease in their fecal Cu excretion, as compared to rats fed the adequate-Cu diet. Previous research using the isotope dilution method also found higher percentages of Cu absorption, and lower fecal Cu excretion, in rats fed a range of low-Cu diets compared to CU-adequate controls (10,13). In the study by Johnson (13), rats fed a diet containing 1.7 ~tg Cu /g diet (a concentration of Cu in the diet similar to our study) elicited a relative Cu absorption of 40%, with endogenous fecal Cu losses of 4 ~tg, approximating the values obtained in our animals.

Although no statistically significant differences were apparent between Zucker and SD rats regarding the percentage of Cu absorption, the degree of adaptive change in the two strains was quite different. Cu absorption was 57% in the SD Low group and 18% in the SD Cont group, more than a 215% increase for the SD Low group. Zucker rats demonstrated a much smaller adaptive response: 39.5% for the Zucker Low and 28.5% for the Zucker Cont groups, a relative increase of only 38%. Thus, compared to Zucker rats, SD rats have an apparently larger capacity to adapt to a low-Cu diet by absorbing more dietary Cu.

An alternative perspective is to examine the effects of dietary Cu on Cu balance if adaptive changes would not have occurred in rats receiving the low-Cu diet. The SD Low rats would have had a net Cu absorption of only 4.2 ~tg if their relative Cu absorption had not increased. This is compared to the 13.1 ~tg of Cu that they actually absorbed, a difference of nearly 9 ~tg. In contrast, the adaptive change in the Zucker strain cre- ated a net increase in Cu absorption of only 2.2 ~g. This nearly fourfold difference between strains suggests that Zucker animals have a lowered adaptive absorptive response to the low-Cu diet compared to SD rats.

Consistent with findings by Johnson and Lee (10), we observed that Cu excretion plays a larger role in maintaining Cu homeostasis than mod- ifications in Cu absorption. In our stud~ the change in relative absorption increased net Cu absorption by only 8.9 and 2.2 ~tg for the SD Low and the Zucker Low groups, respectively. In comparison, the reduction in Cu excretion resulted in a conservation of 29.4 and 19.3 ~g of Cu compared to control diets for the SD Low and the Zucker Low groups, respectively.


276 Mil ler e t al.

Table 4 Effect of D i e t a r y C u o n Tissue C u C o n c e n t r a t i o n s in f i p r a g u e - D a w l e y

a n d Lean Z u c k e r Rats ( F a / F a )

Cu (mmol/g tissue)

Groups

Tissues SD Cont SD Low Zucker Cont Zucker Low

Bone 44.6 +_ 2.05 41,I + 1.58 46.9 + 4.10 38.3 + 5.04

Brain 34.8 + 1.58 30.7 + 1.42 34.2 + 4.10 30.1 + 3.47

Heart 67.4 _ 2.05 a 55.0 + 4.25 ab 65.0 + 3.47 ab 52.9 + 3.15 b

Kidney 84.9 + 8.03 a 43.2 + 6.14 b 82.2 + 7.25 a 43.5 + 4.88 b

Liver 52.6 + 4.73 a 29.8 + 4.73 b 49.8 + 1,73 a 23.0 + 2.99 b

Muscle 22.5 + 3.62 a 14.6 + 2.68 b 21.3 + 4.10 a 10.2 + 3.62 b

Plasma 21.3 + 2.84 a 4.9 + 2.99 b 15.6 + 2.21 a 1.6 + 0.47 b

RBC 11.7 + 1.73 a 4.7 + 0.95 b 9.0 + 0.32 a 5.2 + 0.32 b

Spleen 22.7 + 1.58 a 12.3 + 1.42 b 22.1 + 4.25 a 11.3 + 2.21 b

ANOVA, p value

Diet (D)

Strain (S)

D X $

NS

NS

NS

NS

NS

NS

0.0032

NS

NS

0.0002

NS

NS

0.0001

NS

NS

0.0216

NS

NS

0.0001

NS

NS

0.0004

NS

NS

0.0015

NS

NS

Results are means _+ SEM. Means with different superscripts within a row are significantly different (p _< 0.05) from

each other. Rats were fed a diet adequate in Cu (Cont) or low in Cu (Low).



The isotope dilution method has been utilized to examine absorption and endogenous excretion of Cu and Zn (9,10,13). In this procedure, animals are fed their experimental diet for a minimum of 20 to 24 d (14 d of diet adaptation before isotope administration, and 6-10 d after injection to allow for achievement of isotopic steady-state following 67Cu injection). Results obtained for absorption and excretion of the trace elements do not represent what is occurring immediately following introduction of the diet. Instead, the method provides a description of changes following achievement of a metabolic and isotopic steady-state. Based on our earlier findings, the differences in the rate of adaptation to the low-Cu diet among Zuckers and SD rats may be responsible for the differences in plasma Cu concentrations, liver Cu concentrations, and liver CuZn SOD activity. In our preliminary studies (Miller, unpublished data), we demonstrated that plasma Cp activity in lean Zucker rats decreased 90% of baseline values in 4 d while on a marginal Cu diet (1.1 ~tg Cu/g diet). In a separate study (1), a less severe Cu-deficient diet (3.0 ~tg Cu/g diet) resulted in a 35% reduction in plasma Cp activity for SD rats compared to an 82% decrease in lean Zucker rats. These findings are consistent with the suggestion that SD and obese Zucker rats adapt more quickly to a low-Cu diet than do lean Zuckers. Thus, measurements of Cu uptake, excretion, and Cu status may be significantly different between Zucker and SD strains during the initial stages of dietary introduction. However, as the duration on the low-Cu diet is prolonged, adaptations in Cu homeostasis occur in both Zucker and SD rats such that differences in Cu metabolism between the strains are no longer evident.

Results from the distribution of nonisotopic Cu and 67Cu would pro- vide insight in determining if uptake/storage of Cu is abnormally high in extrahepatic tissues of the Zucker rat. In this scenario, Cu absorption and excretion may be similar for Zucker and SD rats. However, if tissue distribution of Cu is altered, this may, in part, be responsible for the lower plasma and liver Cu concentrations in Zucker vs SD rats observed in earlier studies. In general, the low-Cu diet used in this study significantly reduced nonisotopic Cu concentrations in tissues sampled, except bone and brain. No strain differences in tissue concentrations of Cu were apparent. The largest dietary effect occurred in the plasma, which showed an 83% reduction compared to adequate-Cu groups. A 40-60% reduction in Cu concentration was observed for kidney, liver, muscle, RBC, and spleen, whereas the heart had only a 20% decrease. These reductions are similar to those observed by others (14-16).

An index of shorter-term Cu metabolism is established from tissue 67Cu uptake/retention findings. These results indicated that tissues from the low-Cu groups displayed a higher radioactivity compared to the adequate-Cu groups. However, no differences between strains were apparent. The specific activity reflects the increase in tissue 67Cu retention and the decrease in nonisotopic concentrations in the low-Cu groups.


278 Miller et al.

However, an opposite effect was observed in the heart and plasma, both of which showed an increase in radioactivity (expressed either as ~tCi/g tissue or percent distribution) in the adequate-Cu groups (p = 0.071 for heart nCi /g tissue). In previous research, oral and parenteral administration of 67Cu to Cu-adequate and Cu-deficient rats resulted in an increased retention of the tracer in Cu-deficient animals (16).

Total daily fecal excretion of nonisotope Cu provides evidence that rats in the two dietary groups had reached a metabolic steady state during the initial 14-d dietary adaptation period (Fig. 4A). Cu content in the feces, for the most part, remained at a constant level during the final 9 d of the experiment, especially for the SD Low and Zucker Low groups. Although the absolute values differed depending on the dietary group, the shape of the curves was similar between the groups. This is in contrast to the pattern shown for the fecal excretion of 67Cu (Fig. 3A). Throughout the first 4 d, the curve for control rats had a negative slope, whereas the curve for Cu-deficient rats showed a positive slope. For days 5-9, the shapes of the curve were similar among groups with a plateau or a slow decrease in activity occurring over this period. Confounding these results is the interaction of isotope excretion along with the diffu- sion of the 67Cu dose from the site of injection. The findings suggest that the rates of mobilization from the injection site, utilization of the trace element, and/or subsequent incorporation of 67Cu into compartments for fecal excretion were higher for the control groups than for low-Cu groups. It is proposed that the time required to achieve a plateau for 67Cu activity in feces is going to be prolonged in animals fed a low-Cu diet compared to those fed an adequate-Cu diet. For the control groups, the higher initial activity is due to a faster rate of the equilibration of the trace mineral into body pools concerned with its excretion. In contrast, Cu-deficient animals attempt to conserve the Cu. Therefore, it takes longer before the 67Cu dose is excreted. This point is further emphasized in Fig. 6, which illustrates that the control groups achieved isotopic steady state soon after injection (by day 2), whereas Cu-deficient rats do not reach this plateau for several days.

This investigation has confirmed earlier findings regarding Cu absorption and endogenous excretion in rats maintained on a low-Cu diet. No absolute differences in percentage of Cu absorption or endogenous fecal excretion were seen among Zucker and SD rats. However, the relative changes in the percentage of Cu absorption in response to a low- Cu diet appear to be more marked for SD rats. Thus, a different experimental method to detect the dynamic changes in Cu absorption or endogenous excretion occurring soon after diet initiation may aid in the understanding of the Cu aberration in the lean Zucker rat. Further studies in this area are needed to compare the time-course of adaptive responses, which occur m rats fed a low-Cu diet.



ACKNOWLEDGMENT

This work was supported in part by the National Institutes of Health through HD-26777 and DK35747.

REFERENCES

1. C. L. Keen, K. L. Olin, M. H. Oster, D. C. Thurmond, B. J. German, J. S. Stern, and S. D. Phinney, The obese Zucker rats (fa/fa) and its lean control (Fa/Fa) are charac- terized by marked differences in the antioxidant defense system, The FASEB J. 6, A1677 (abstract) (1992).

2. S. M. Lynch and L. M. Klevay, Contrasting effects of a dietary copper deficiency in male and female mice, Proc. Soc. Exp. Bio. Med. 205, 190-196 (1994).

3. M. Suzuki and T. Aoki, Impaired hepatic copper homeostasis in Long-Evans Cinnamon rats: reduced biliary excretion of copper, Ped. Res. 35, 598-60t (1994).

4. N. Sugawara, M. Katakura, D. Li, C. Sugawara, and H. Miyake, Role of hepatic copper-metallothionein on liver function of Long-Evans Cinnamon rats with a new mutation causing hereditary hepatitis, Res. Commun. Chem. Pathol. Pharm. 83, 349-358 (1993).

5. H. Nederbragt, Strain and sex-dependent differences in response to a single high dose of copper in the rat. Comp. Biochem. Physiol. 81C, 425-431 (1985).

6. H. Nederbragt and A. J. Lagerwerf, Strain-related patterns of biliary excretion and hepatic distribution of copper in the rat, Hepatology, 6, 601-607 (1986).

7. M. Fields, C. G. Lewis, M. Lure, and W. E. Antholine, The influence of gender on developing copper deficiency and on free radical generation of rats fed a fructose diet, Metabolism, 41, 989-994 (1992).

8. M. C. Linder, P. A. Houle, E. Isaacs, J. R. Moor, and L. E. Scott, Copper regulation of ceruloplasmin in copper-deficient rats, Enzyme 24, 223-235 (1979).

9. E. Weigand and M. Kirchgessner, Radioisotope dilution technique for determination of zinc absorption in vivo, Nutr. Metab. 20, 307-313 (1976).

10. P. E. Johnson and D. Y. Lee, Copper absorption and excretion measured by two methods in rats fed varying concentrations of dietary copper, J. Trace Elements Exp. Med. 1, 129-141 (1988).

11. M. S. Clegg, C. L. Keen, B. Lonnerdal and L. S. Hurley, Influence of ashing techniques on the analysis of trace elements in animal tissue. I. Wet ashing, Biol. Trace Element Res. 3, 107-115 (1981).

12. C. A. Owen, Distribution of copper in the rat, Am. J. Physiol. 207, 446-448 (1964). 13. P. E. Johnson, Effect of various dietary carbohydrates on absorption and excretion of

copper in the rat as measured by isotope dilution, J. Trace Elements Exp. Med. 1, 143-155 (1988).

14. L. Rossi, M. R. Ciriolo, E. Marchese, A. DeMartino, M. Giorgi, and G. Rotilio, Differ- ential decrease of copper content and of copper binding to superoxide dismutase in liver, heart and brain of copper-deficient rats, Biochem. Biophys. Res. Commun. 203, 1028-1034 (1994).

15. D. A. Schuschke, J. T. Saari, J. W. Nuss, and F. N. Miller, Platelet thrombus formation and hemostasis are delayed in the microcirculation of copper-deficient rats, J. Nutr. 124, 1258-1264 (1994).

16. C. A. Owen, Metabolism of copper 67 by the copper-deficient rat, Am. ]. Physiol. 221, 1722-1727 (1971).


copper absorption, endogenous excretion, and distribution in sprague-dawley and lean (fa/fa) zucker...

Documents