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Department of Nutritional Science, Tokyo University of Agriculture, 1–1 Sakuragaoka 1, Setagaya, Tokyo 156-8502;
* Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 263-8522;
Department of Pathology, School of Medicine, Akita University; and
** Biochemistry Division, National Cancer Center Research Institute, 1–1, Tsukiji 5, Chuo-ku, Tokyo 104-0045, Japan
2To whom correspondence should be addressed. E-mail: mnagao{at}nodai.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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80% higher than in rats fed the control diet. Liver iron concentrations did not differ among the groups. The results, including histological analyses, indicate that SPI enhances copper uptake into the liver cells and promotes liver cell damage in LEC rats. However, this did not occur in the livers of F344 rats with wild-type Atp7b. Recommendations to individuals suffering from Wilson’s disease to avoid consuming soy protein may be warranted.
KEY WORDS: • LEC rats • soy protein isolate • copper • Wilson’s disease • liver cell damage
Recently, soy has attracted a great deal of attention as a health food, and epidemiologic studies have indicated that consumption of soy products reduces the risk of breast and other cancers (1
). Soy protein isolate (SPI), with or without isoflavone supplementation, is frequently consumed and is well balanced in necessary amino acids. It has also been demonstrated that the intake of soy protein enhances the excretion and reduces the serum concentration of cholesterol (2
). Soy protein is also given as a milk alternative to infants who are allergic to cow’s milk protein, or who suffer from post-diarrhea lactose intolerance, galactosemia or primary lactase deficiency. Although the American Academy of Pediatrics recommends breastfeeding over soy formula (3
), at present, 25% of the 4 million babies born each year in the United States are fed soy infant formula (4
).
Hereditary Wilson’s disease, due to a defect in the ATP7B gene (5
–7
) encoding a protein involved in the transport of copper from the liver to the bile and blood, affects 30 per million people across the globe. The disease involves liver cell damage caused by the accumulation of copper. The Long-Evans rat with a cinnamon-like coat color (LEC rat) also has a deletion mutation at the carboxyl terminal of the Atp7b gene so that ATP binding activity is lost (8
). As a result, LEC rats have a defect in the transport of copper from the liver to the bile and blood, and suffer from liver cell damage with jaundice at 16–20 wk of age caused by accumulation of the metal, presumably mediated by reactive oxygen species (9
). The model is therefore appropriate for examination of possible modifiers of Wilson’s disease. Chelators such as D-penicillamine, trienthine and tetrathiomolybdate are used to treat people with Wilson’s disease (10
–12
). It is also important to reduce the daily load of copper from food. However, it is not known whether there are food factors that influence hepatic copper metabolism.
In the present study, SPI, defatted soy (DFS) and SPI supplemented with L-methionine (SPIM) were administered in the diet to LEC rats to determine effects on the development of liver cell damage and jaundice in relation to hepatic copper concentration.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Guidelines established by our institutional animal Care and Use Committee were followed for all procedures. The protocol for the present study was reviewed and approved by the TUA Animal Care and Use Committee of our university. LEC and F344 male rats were purchased from Charles River Japan (Yokohama, Japan) and CLEA Japan (Tokyo, Japan), respectively, at the age of 5 wk. They were housed in individual wire cages in a ventilated and temperature-controlled room at 22 ± 1°C with 60–65% humidity and a 12-h light:dark cycle. Rats consumed the AIN-93G diet (13 ) and deionized water ad libitum.
Diets.
Each component of the AIN-93G (control) diet was purchased from Dyets (Bethlehem, PA) and prepared according to the AIN formulation (13 ). SPI and DFS, kindly provided by Fuji Oil (Osaka, Japan), were prepared from nongenetically modified soy imported from the United States (IOM grade, produced in Iowa, Ohio or Minnesota) by extraction with n-hexane of the cotyledon meal after removal of hulls and hypocotyls (DFS), and then by extraction with hot water under neutral conditions, acid precipitation, neutralization, sterilization and drying (SPI) (14 ). For the SPI diet, casein (200 g/kg) in the control diet was replaced by SPI, composed of the following (g/100 g): protein, 85.6; dietary fiber, 4.2; fatty acids, 1.6; water, 5.7; and ash, 4.6, with copper and iron concentrations of 12 and 73.2 µg/g, respectively. The amounts of cupric carbonate and ferric citrate in the mineral mix were adjusted to give the same final concentration as in the control diet. For the DFS diet, DFS, containing (g/100 g) protein, 45.8; carbohydrate, 30.8; water, 11.9; ash, 5.5; and fat, 2.9 was added to make 22 g/100 g of the DFS diet; the amounts of casein and cornstarch were reduced to 8.0 and 29.8 g/100 g diet. The composition is shown in Table 1. The copper and iron concentrations were 7.8 and 36.4 µg/g, respectively, and the amounts of cupric carbonate and ferric citrate in the mineral mix were adjusted as needed. The SPIM diet was prepared by adding L-methionine to the SPI diet at a concentration of 1.8 g/kg to equal the methionine concentration of the control diet. Copper and iron concentrations in samples from control, SPI and DFS diets were measured by absorption spectrophotometry after being dry-ashed at 550°C. Copper concentrations were 6.0 ± 0.3, 6.0 ± 0.2 and 6.4 ± 0.3 µg/g (mean ± SD, n = 3), respectively, and iron concentrations were 35 ± 2, 36 ± 3 and 36 ± 1 µg/g, respectively.
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In Experiment 1, LEC male rats (n = 10), consumed the control, SPI or DFS diet ad libitum beginning at 6 wk of age. In Experiment 2, LEC male rats (n = 10) consumed the control or SPI diet ad libitum beginning at 6 wk of age. In Experiment 3, LEC male rats (n = 10) consumed the control, SPI or SPIM diet ad libitum beginning at 6 wk of age. In all 3 experiments, when rats became moribund with jaundice, they were euthanized while under ether anesthesia. In Experiment 3, blood was collected from the cervical aorta; serum was prepared and stored at -80°C until use. Livers were removed and fixed in 40 g/L buffered paraformaldehyde.
Serum biochemistry (Expts. 3 and 4).
In Experiment 4, Fischer male rats (n = 5) consumed the control or SPI diet ad libitum beginning at 6 wk of age and they were killed at the age of 10 wk while under anesthesia. Collected serum samples and livers were quickly frozen and stored at -80°C until use. Concentrations of iron (15 ), total protein (16 ), albumin (17 ), ferritin (18 ), total cholesterol (19 ), LDL cholesterol (20 ), HDL cholesterol (21 ), triglycerides (22 ) and bilirubin (23 ), the ratio of albumin to globulin (24 ), and activities of aspartate aminotransferase (AST) (25 ), alanine aminotransferase (ALT) (26 ), alkaline phosphatase (27 ) and leucine aminopeptidase (28 ) were measured in all rats at 10 wk of age (Expt. 4). Serum bilirubin concentration and activities of AST and ALT were determined in five randomly selected moribund rats (Expt. 3).
Liver metal concentrations (Expts. 3 and 4).
Liver homogenate (200 µL of a 200 g/L preparation) in 50 mmol/L Tris-HCl and 250 mmol/L glucose (pH8.0) was wet-ashed with an acid mixture (HNO3/HClO4, 4:1, v/v), and quantified using an inductively coupled plasma-mass spectrometer (Agilent Technologies, Musashino, Japan).
Histochemical staining (Expts. 3 and 4).
Livers fixed in 4% buffered paraformaldehyde were routinely processed, sectioned and subjected to routine hematoxylin and eosin (H-E) staining. Copper staining was performed by the sulfide-silver method (29 )
Statistical analysis.
Data are presented as means ± SD. The significance of difference between the control and the experimental groups was assessed using the nonparametric Mann-Whitney test, and significance of intergroup differences by the nonparametric Kruskal-Wallis test. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Food intakes did not differ among groups at wk 10. Body weights did not differ among groups through wk 9 (Expt. 1, Fig. 1 ). The body weights of the DFS and SPI diet groups differed from that of controls beginning at wk 10 and 11, respectively. Control rats became moribund between the ages of 16 and 21 wk, with symptoms of jaundice. The symptoms included a decrease in body weight, yellowish skin, ears, tail and genital region, hematuria, oliguria, subcutaneous hemorrhage and sluggish movement. The mean survival time of controls was 19.1 ± 1.7 wk (Fig 2. ). In contrast, rats fed SPI became moribund with the same symptoms between the ages of 12 and 15 wk with a shorter survival time of 14.0 ± 0.8 wk (P < 0.001; Expt. 1). The survival time of rats in the DFS diet group was intermediate and different from both other groups (P < 0.05). Similar results were obtained in Experiment 2. Survival times were 21.4 ± 2.9 wk for controls and 16.1 ± 1.3 wk for the SPI diet group (P < 0.001). In Experiment 3, the survival times of the SPI (14.2 ± 1.0) and SPIM diet groups (14.9 ± 1.0) did not differ. When the control and SPI diet groups in Experiments 1–3 were pooled, survival times were 20.6 ± 3.3 and 14.7 ± 1.4 wk, respectively.
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Liver copper concentration in the control group was within the range reported for LEC rats [1.57 ± 0.35 µmol/g (n = 4)]. In the SPI and SPIM diet groups, the copper concentrations were 80% greater (P < 0.05; Table 2). Liver iron concentrations did not differ among groups, but zinc concentrations in the SPI and SPIM diet groups were significantly higher than that in the control group (Table 2).
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The 14 biochemical markers examined did not differ among groups at the age of 10 wk or when rats became moribund (data not shown).
Histological analysis (Expts. 3 and 4).
H-E staining revealed almost no liver cell damage in control (Fig. 3a ), SPI- (Fig. 3 c) or SPIM-fed (data not shown) LEC rats at 10 wk. No evidence of toxicity of the SPI diet was observed in F344 rats at 10 wk (Fig. 3 e). Copper staining demonstrated accumulation of this metal in hepatocytes of LEC rats. Staining was weaker in LEC rats fed the control diet (Fig. 3 b) than in those fed the SPI diet (Fig. 3 d) or the SPIM diet (data not shown). There was no copper staining in the livers of F344 rats at the age of 10 wk (Fig. 3 f).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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A characteristic property of soy protein is that it is low in methionine, although the level in SPI is considered to be sufficient for humans even if soy is their sole source of dietary protein (13 ,30 –32 ). Because rats require large amounts of methionine to maintain hair growth, we could not exclude the possibility that a deficit of methionine resulted in an insufficient supply of methyl groups to cells and consequently enhanced cell damage induced by copper. However, L-methionine supplementation did not increase the survival time of LEC rats fed SPI.
To assess the possibility that impairment of detoxification mechanisms might have played a role, we also examined the levels of reduced and oxidized glutathione in the livers of rats in the control and SPI diet groups at 10 wk of age, but there were no differences between groups (33 ). Metallothionein, which is necessary for copper detoxification, was also expressed similarly in the livers of rats in the control and SPI diet groups at 10 wk of age (33 ). Thus, the liver cell damage due to the SPI diet may be due to the increase in hepatic copper concentration, which presumably was due to greater uptake.
Iron also has been implicated in the pathogenesis of LEC rats, whose liver iron concentration is 2.3 times that of normal rats. Further, reduction of dietary iron prolongs survival (34
). Phytic acid, present at a high concentration of 2 g/100 g in SPI, suppresses uptake of iron in the liver (35
,36
). However, in the present study, hepatic iron concentrations were the same in the control and SPI diet groups. Because phytic acid apparently does not affect copper uptake (36
), it presumably did not play an important role in copper accumulation. In addition, zinc concentrations in the livers of rats in the SPI and SPIM diet groups were elevated, although phytic acid suppresses zinc absorption (35
,36
).
Healthy F344 rats with the wild-type Atp7b gene showed no increase in copper levels in the liver at the age 10 wk, despite consuming the SPI diet. Although it is plausible that the different effect of the SPI diet on LEC and F344 rats is due to the Atp7b protein, possible involvement of other gene product(s) cannot be ruled out. At present, it is not known whether copper was taken up into F344 rat hepatocytes at the same high rate as in LEC rats. Because copper is taken up by cells with the aid of copper transporter protein (CTR1) after reduction to a cuprous ion (37 –39 ), studies of SPI diet effects on Ctr1 expression in LEC and F344 rats should provide insight into the underlying mechanisms. Copper is found in various enzymes such as ceruloplasmin, cytochrome oxidase, superoxide dismutase, dopamine ß-hydroxylase, lysyl oxidase, coagglutination factor V and VII, and hephaestin; therefore, it plays an important role in physiology (40 ). Because soy contains a high level of copper, it is a good source for normal individuals. However, from the present results, it cannot be recommended for sufferers from Wilson’s disease not only because of high copper concentration, but also because of the presence of some factor(s) that enhance liver cell copper uptake.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: ALT, alanine aminotransferase; AST, aspartate aminotransferase; DFS, defatted soy; H-E, hematoxylin and eosin; LEC rat, Long-Evans rat with a cinnamon-like coat color; SPI, soy protein isolate; SPIM, SPI with L-methionine supplement.
Manuscript received 7 October 2002. Initial review completed 17 November 2002. Revision accepted 29 January 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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