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Northern Ireland Centre for Diet and Health, University of Ulster at Coleraine, BT52 ISA, Northern Ireland
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • diamine oxidase • copper status • cuproenzymes • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Plasma copper and ceruloplasmin concentrations, along
with the enzyme activity of ceruloplasmin oxidase (EC 1.16.3.11), may
not respond to small manipulations of dietary copper and may not be
sensitive enough to detect suboptimal copper status (Hopkins and Failla 1995
). Ceruloplasmin is also an acute phase protein and
is increased during inflammation, which could further mask an
underlying marginal copper deficiency (DiSilvestro 1990
,
DiSilvestro and Marten 1990
).
Activity of CuZn-superoxide dismutase
(SOD)3
is reported to decrease in rats fed both copper-deficient and
copper-marginal diets (DiSilvestro et al. 1997
).
Changes in the activity of SOD are organ specific, and liver SOD
activity was the most sensitive to deficiency of all of the organs
examined by Paynter et al. (1979)
. The decrease in organ
SOD activity is accompanied by a drop in erythrocyte SOD activity,
which appears to be relatively sensitive to copper deficiency
(Bettger et al. 1978
).
Several other investigators report that organ cytochrome
c oxidase (CCO) activity responds to a decrease in
dietary copper intake by a subsequent drop in organ enzymatic activity
(Prohaska 1990
). Platelet CCO activity, which reflects
the alterations in hepatic CCO activity, has been investigated as an
indicator of copper status (Johnson et al. 1993
), and
changes in CCO activity are thought to be more sensitive to changes in
copper status than SOD activity (Prohaska 1991
).
Diamine oxidase is a copper-dependent enzyme responsible for the
oxidative deamination of diamines such as cadaverine, putrescine, some
of their derivatives and histamine (Wolvekamp and DeBruin 1994
). Plasma diamine oxidase activity may be sensitive to
changes in copper status; it was reported recently that this activity
is very low in rats fed a copper-marginal diet for a period of 6
mo, and significantly different from that in controls fed a
copper-adequate diet (DiSilvestro et al. 1997
). The
objective of this study was to investigate differences in activity of
this enzyme compared with other copper-dependent enzymes in rats
fed copper-adequate, copper-marginal and copper-deficient
diets.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male weanling Sprague-Dawley rats (n = 21;
in-house colony), ~21 d old, were randomly assigned to one of
three dietary regimens, copper deficient (CuD), n = 7; copper marginal (CuM), n = 7; or copper adequate
(CuA), n = 7. The diets used were based on a
modification of the recommendations of the American Institute of
Nutrition (AIN 1977
) and contained the following
major components (g/kg diet): sucrose, 400; casein, 200; cornstarch,
150; corn oil, 150; cellulose, 50, modified AIN-76 mineral mix, 35;
AIN-76 vitamin mix, 10; DL-methionine, 3; choline
bitartrate, 2. Cupric carbonate was omitted from the AIN-76 mineral mix
for the CuD diet, and was added at 0.1 and 0.3 g/kg of the mineral mix
in the preparation of the CuM and CuA diets, respectively. Each diet
was analyzed for copper content by atomic absorption spectroscopy. Rats
had free access to both food and deionized water for 57 d. Food
was provided freshly every day and deionized water twice weekly. Food
intake was recorded daily and rat body weights were recorded weekly.
Each rat was housed separately in a metabolic cage, in a temperature-
and humidity-controlled environment, with a 12-h light:dark cycle.
This experiment received approval from the Department of Health and
Social Services under the Animals (Scientific Procedures) Act 1986.
Sample collection.
On completion of the experiment, all rats were weighed and anesthetized under isoflurane (Abbott Laboratories, Queenborough, Kent, UK). Upon anesthesia, blood was drawn via cardiac puncture and immediately transferred into heparinized blood tubes. Blood was centrifuged at 1800 x g for 10 min at 25°C (Mistral 2000, MSE Scientific,Crawley,Sussex,UK) and plasma divided into aliquots and frozen at -80°C until analysis. The remaining erythrocyte pellet was resuspended to the original blood volume with PBS (pH 7.4), washed three times in PBS at 200 x g for 10 min at 25°C, divided into aliquots and stored at -80°C until analysis. After the rats were killed by cervical dislocation, heart, liver and kidneys were removed and placed immediately in 0.25 mol/L sucrose buffer [containing Tris (5 mmol/L), EDTA and disodium salt (1 mmol/L) dissolved in distilled water, adjusted to pH 7.4 with HCl] at 0°C. Organ weights were recorded and livers were divided into portions of equal weight for homogenate preparation and copper content analysis, respectively. All samples were placed in plastic vials (Sterlin, Feltham, UK) and stored at -80°C until analysis.
Liver and heart homogenate preparations were performed on the day of enzyme analysis. After being thawed on ice, tissues were homogenized to a 250 g/L solution in 0.25 mol/L sucrose buffer using an Ultra-Turrax homogenizer (Janke and Kunkel GMBH and IKA-Labortechnik, Straufen, Germany). Homogenates were then centrifuged at 800 x g for 10 min at 4°C, the supernatant removed and the pellet resuspended in 0.25 mol/L sucrose buffer to a final concentration of 250 g/L. No further sample dilution was required on homogenates before CCO determination. Homogenates were diluted to a final concentration of 0.5 g/L for analysis of SOD activity. Further dilution of homogenates was required for individual samples with enzyme activities beyond test detection range.
Chemical analyses.
Samples of each batch of diet (5 ± 0.1 g) were ashed at 555°C, the residue digested in 25 mL of 8.3 mol/L HCl (BDH Chemicals, Poole, Dorset, UK) by boiling until a clear yellow solution was obtained. Samples were then filtered through no. 41 Whatman filter paper and diluted with deionized water to 50 mL. Blanks (25 mL) of 8.3 mol/L HCl were prepared similarly to samples, and copper measured with a atomic absorption spectrophotometer (Pye Unicam SP9, Cambridge, UK) against standards set between 0.25–5.0 mg/L (Spectrosol, BDH Chemicals).
Liver copper concentrations were analyzed by atomic absorption spectrophotometry and expressed on a dry weight basis. Liver samples were thawed, dried to a constant weight in a hot air oven (Gallenkamp, ABC Scientific, Carrickfergus, UK) at 105°C, and digested in 5 mL concentrated HNO3 for 24–48 h. Blanks of 5 mL concentrated HNO3 were processed in conjunction with the samples. Upon cooling, samples and blanks were diluted with 10 mL of deionized water, filtered through no.1 Whatman filter paper, and further diluted to 20 mL with distilled water. Blanks and samples were analyzed on a AA-6701/6601 Atomic Absorption Spectrophotometer (Shimadzu, Diusberg, Germany). A copper atomic absorption standard solution (Sigma, Aldrich Chemical, Poole, Dorset, UK) was used to prepare a standard curve from 0 to 4 mg/L. A quality control sample was prepared by using an assayed pooled serum that had a designated copper concentration (Randox Laboratories, Crumlin, Antrim, UK).
Enzymatic analyses.
Ceruloplasmin oxidase activity was determined by a modification of the
method described by Henry et al. (1960)
, using
p-phenylenediamine dihydrochloride (PPD; Sigma,
Aldrich) as substrate and measuring the rate of oxidation of PPD·2HCl
at 37°C. Analysis was performed on the Cobas Fara automated analyzer
(Roche, Basel, Switzerland).
Total and CuZn-SOD activity was determined in erythrocyte, livers
and hearts using a previously described (Brown and Strain, 1990
) modification of the method of Jones and Suttle (1981)
with a commercially available kit (RANSOD) (Randox).
Analysis was carried out on the Cobas Fara automated analyzer (Roche)
at 500 nm. Activity of SOD in erythrocytes was expressed per gram of
hemoglobin, and in hearts and livers per gram of protein.
The hemoglobin concentration was determined by diluting erythrocytes (40 µL) with 20 mL of ISOTON (Coulter Electronics, Bedfordshire, UK) in a plastic vial (Sterilin, BDH Chemicals). The samples (g/L) were measured on a hemoglobinometer (Coulter Electronics) after the addition of ZAPOGLOBIN (Coulter Electronics).
Protein concentrations in heart and liver extracts were determined by a
modification of the method of Bradford (1976)
using a
commercially available kit (Bio-Rad Laboratories, Hertfordshire, UK).
Protein concentrations were measured against a standard curve prepared
from bovine serum albumin (Sigma, Aldrich).
Liver and heart CCO activities were measured enzymatically on the Cobas
Fara automatic analyzer (Roche) using a modification of the method of
Smith (1955)
. The substrate was prepared by reducing
reconstituted horse heart cytochrome c (Sigma, Aldrich)
(4.3 mg in 3.25 mL of 0.1 mol/L potassium phosphate buffer, pH 7.0)
with excess (10 mg) sodium dithionite (Sigma, Aldrich). Excess sodium
dithionite was removed by gel filtration using a G-25 Sephedex column
(Pharmacia Biotech, Uppsala, Sweden). The final substrate concentration
was standardized at 57.7 mmol/L ± 0.3% to ensure a concentration
of 50 µmol/L in the final reaction mix. This was
achieved by ensuring an optical density at 550 nm of 1.131 ± 0.3%. A ratio of the optical density at 550 and 565 nm >10 was
accepted to ensure sufficient reduction. CCO activity was expressed as
U/mg protein.
Plasma diamine oxidase activity was determined using a modification of
the method of Takagi et al. (1994)
. Analyses was
conducted at 37°C on the Cobas Fara automated analyzer (Roche). A
substrate solution was prepared by dissolving 606.1 mg cadaverine
dihydrochloride (Sigma, Aldrich) in 100 mL of 25 mmol/L PIPES buffer
containing 0.5% Triton X-100 (pH 7.2), and 130 µL was
incubated for 1 min at 37°C. Plasma and standards (30
µL) were added with 20 µL PIPES
dilution buffer and the reaction was incubated for a further 30 min.
The development of methylene blue was initiated by adding 150
µL of color solution containing
10-(carboxymethylaminocarbonyl)-3,7-bis (dimethylamino) phenothiazine
sodium salt (Wako Chemical, Osaka, Japan), ascorbate oxidase and
peroxidase type X horseradish (Sigma Chemical) to the reaction mix; the
rate of color development was measured at 668 nm over 12 min and
quantified (U/L) by a standard curve prepared using diamine oxidase
(Sigma, Aldrich).
Statistical analysis.
All data were analyzed using the SPSS 6.1 for Windows (Chicago, IL) statistical package. Significant differences between the separate treatment groups were analyzed by ANOVA with tests for least significant difference. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rats fed the three diets had significantly different liver copper
concentrations (Table 1
). Liver and heart SOD and CCO activities were lower in the CuD rats
compared with both CuA and CuM rats. The differences were accompanied
in CuD rats by lower plasma ceruloplasmin and erythrocyte SOD
activities than in the other two dietary groups. Plasma diamine oxidase
activity was significantly higher in CuA rats compared with both CuM
and CuD rats (Table 1)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Medeiros et al. 1993
, Prohaska 1990
) compared with groups fed
1.73 or 6.7 mg Cu/kg diet. Traditional enzymatic indicators of copper
status, including liver and heart CCO and SOD activities, were also
depressed in these rats compared with the other two groups. The
decrease in organ cuproenzyme activity was reflected in the response of
circulating indicators of copper status such as decreased serum
ceruloplasmin oxidase and erythrocyte SOD activities in the CuD rats
compared with the two other groups.
Consistent with other studies (Allen 1996
,
Al-Othman et al. 1992
, Fields and Lewis 1997
), rats fed a CuD diet had a lower body mass and a reduced
food intake throughout the trial. This phenomenon is reported to occur
independently of the fat or carbohydrate composition of the CuD diet
provided (Fields et al. 1996
, Jalili et al. 1996
). Rats with access to the CuM diet were significantly
heavier, compared with the CuA- and CuD-fed rats at the end of the
study. The reason behind this is unclear because it was not accompanied
by increased food intake. Liver copper decreases in rats after only 1
wk of consuming a marginally low copper diet (Schuschke et al. 1995
), and in this study, differences in hepatic copper
concentrations characterized the three dietary groups.
Traditional enzymatic indicators of copper status were not affected by
the CuM diet containing 1.73 mg Cu/kg diet. The limited effect of a CuM
diet on traditional indicators of copper status has been demonstrated
before (Hopkins and Failla 1995
). In that study, a CuM
diet of 2.8 mg Cu/kg diet was consumed for >6 mo without any adverse
effects on copper indices.
Results from this study suggest that a CuM diet of 1.73 mg/kg diet can
be consumed by rats for at least 8 wk without any deleterious responses
in many of the copper indices measured. Notably, hepatic copper
concentrations, final body mass and serum diamine oxidase activity were
the only physiologic or biochemical variables that differed between the
CuM and CuA rats. The observation that plasma diamine oxidase activity
was lower in marginally copper-deficient rats is in agreement with
a previous study (DiSilvestro et al. 1997
). In that
study, however, a diet marginally deficient in copper (2 mg Cu/kg diet)
was supplied for 6 mo; it not only depressed plasma diamine oxidase
activity but also lowered plasma ceruloplasmin and liver SOD
activities. We observed that alterations in plasma diamine oxidase
activity occurred in marginally copper-deficient rats before any
adverse effects were detected in other cuproenzymes, and that plasma
diamine oxidase activity was also low in severely copper-deficient
rats.
The results of this study suggest that rats fed a CuD diet for 8 wk exhibited classical signs of copper deficiency, even though their CuM-fed counterparts did not. The traditional enzymatic methods of evaluating copper status can distinguish between CuA and CuD rats, but often cannot distinguish between CuA and CuM rats. Hepatic copper concentration and plasma diamine oxidase activity alone were sufficiently sensitive to differentiate CuM from CuA rats. These results indicate that plasma diamine oxidase activity may be more sensitive to changes in copper status than the activities of other readily measured copper metalloenzymes.
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FOOTNOTES |
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3 Abbreviations used: CCO, cytochrome c oxidase; CuA, copper-adequate diet; CuD,
copper-deficient diet; CuM, copper-marginal diet; PPD,
p-phenylenediamine dihydrochloride; SOD, Cu-Zn superoxide dismutase.
Manuscript received February 25, 1999. Revision accepted August 25, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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