Department of Chemistry and Biochemistry, and Institute for Molecular Biology and Nutrition, California State University, Fullerton, California 92834-6866
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ABSTRACT |
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The delivery of copper to mammary gland and milk and the effects of lactation were examined in rats. Traces of 67Cu/64Cu(II) were injected intraperitoneally or intravenously into virgin rats or lactating rats (2-5 days postpartum), and incorporation into blood, milk, and tissues was monitored. In virgin rats, most of the isotope first entered the liver and kidney. In lactating rats, almost 60% went directly to the mammary gland. Uptake rates and copper contents of the mammary gland were 20-fold higher in lactation. 67Cu/64Cu appeared in milk and milk ceruloplasmin as rapidly as in mammary tissue and when there was no 67Cu/64Cu-ceruloplasmin in the maternal plasma. Plasma 125I-labeled albumin entered milk much more slowly. Milk ceruloplasmin (10 mg/l) had 25% of the 67Cu/64Cu. Milk copper was 3.3 mg/l. Thus lactation markedly enhances the avidity of the mammary gland for copper, diverting most of it from liver and kidney to that tissue. Also, the primary source of milk ceruloplasmin is the mammary gland and not the maternal plasma.
ceruloplasmin; copper-67; copper-64; iodine-125 albumin; pregnancy; virgin rats
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INTRODUCTION |
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IT IS WELL DOCUMENTED that, during gestation in mammals, the fetus accumulates stores of certain metal ions in the liver. These are used up during the rapid growth of the infant after birth (22, 26). Copper is one of the metal ions to which this observation pertains and where such changes in liver stores have been documented (21, 22, 26). Thus, in most mammalian offspring at birth, the concentration of copper in the liver is higher than at any other time in the normal life of that species (21). In the human, concentrations at birth are ~75 µg/g liver (wet wt); during suckling, they fall to ~20 µg/g; in the normal adult, they are ~5 µg/g (18, 21). In rats, concentrations at birth are ~30 µg/g, compared with 4-5 µg/g in the adult (20, 21). The importance of this fetal copper store has been emphasized since the time of Widdowson et al. (57) because, as they pointed out, the copper content of milk (the initial food of the infant) is relatively low: ~0.25 mg/l in the case of the human, but higher in the case of pigs and rats (50). Nevertheless, studies of the total copper content of rat pups during suckling indicate that they are rapidly accumulating whole body copper (16, 21, 50, 53, 58), which means they must be getting it from the milk. If so, the mammary gland must be actively taking up this trace element and transferring it to the milk, although this has not been specifically examined.
Recent studies on milk from our laboratory have indicated that ceruloplasmin, a copper-binding protein known for its abundance in the blood plasma, is present in the milk of the several mammals so far examined (59). Moreover, we have obtained evidence in rats that the copper in ceruloplasmin, when fed to pups (59), is more readily available for absorption than is nonceruloplasmin copper. Initial studies with piglets indicate that there is a specific ceruloplasmin receptor in the intestinal brush border that disappears after weaning (27). In analogy with the intestinal receptor for lactoferrin, which is thought to provide for specific iron uptake from the milk (10), a ceruloplasmin receptor may allow preferential intestinal uptake of ceruloplasmin copper. A copper transport role for blood plasma ceruloplasmin has been documented by studies in several laboratories, which have shown that ceruloplasmin is a good source (or preferential source) of copper for many tissues (see Ref. 23). This is particularly the case for the placenta and fetus during gestation (19). At the same time, ceruloplasmin is not essential for copper metabolism, in that absorption, tissue distribution, and excretion of copper are not abnormal in the knockout mouse (33). Copper in ceruloplasmin is not dialyzable or exchangeable, and it is thought that delivery from ceruloplasmin must involve specific receptors for this protein that have been detected in the plasma membranes of most cells (21, 49, 51). Other sources of copper in the plasma are albumin and transcuprein, the latter a form of macroglobulin (Liu N, Lo LSL, Goforth J, Vivas E, Sebastian S, Ackary SH, Tsai M, and Linder MC, unpublished observations). These proteins bind Cu(II) when it first enters the blood, as from the diet, and seem to target it to the liver and kidney, where most of the copper is initially deposited (22, 23, 25) or where it is brought for biliary excretion (21).
mRNA for ceruloplasmin has been detected in mammary tissue (15, 28) and in the cells known to produce the milk (15). Its role in providing ceruloplasmin to the milk has been questioned, because ceruloplasmin mRNA is also detected in the mammary gland in the absence of lactation and in male as well as female rats (15). However, we have shown in pigs that mammary gland expression increases markedly at the end of pregnancy and especially in conjunction with lactation (7), and that changes in the levels of mammary gland mRNA expression parallel those of ceruloplasmin production for the milk. Both of these findings imply that milk ceruloplasmin is produced by the mammary gland.
Potential roles for ceruloplasmin have multiplied in recent years. Expression of the protein or its mRNA has been detected in a variety of tissues, including certain cells in the central nervous system and elsewhere, where it also occurs in glycophosphatidylinositol-anchored form (39, 45). Ceruloplasmin has long been recognized as capable of scavenging oxygen radicals (12, 13), which would explain its increased synthesis by the liver during inflammation. An additional role in the exit and entry of iron from/to hepatocytes and other cells is the object of intense current scrutiny on the basis of involvement of another blue copper protein (Fet3) in yeast iron uptake (4, 5, 8); the identification of another potential homolog, hephaestin, in connection with intestinal (and perhaps also placental) iron transport (9, 55); and observations that ceruloplasmin deficiency, due to copper deficiency or mutations in the ceruloplasmin gene, causes iron accumulation (14, 36, 37, 43, 44, 61).
The studies reported here were designed to increase our understanding of copper transport during lactation and the involvement of the mammary gland, by use of the rat as a model. This had not been previously studied. Indeed, we were unable to find reports of even the copper content of mammary tissue. The specific questions addressed were the following. How rapidly does the mammary gland take up copper from the maternal circulation, and how does lactation influence this process? Is plasma ceruloplasmin the preferred source of copper for the mammary gland? How rapidly does copper enter the milk after being absorbed by mammary tissue? Does incorporation of copper into milk ceruloplasmin lag behind the appearance of nonceruloplasmin copper in the milk (i.e., does plasma ceruloplasmin enter the milk in significant amounts)? Our results show that the mammary gland is very active in copper nutrition and metabolism during lactation and is the primary source of ceruloplasmin entering rat milk.
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MATERIALS AND METHODS |
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Rats and treatments.
All studies were preapproved by the Institutional Animal Care and Use
Committee of California State University, Fullerton (protocol nos.
98-R-05 and 01-R-02). Sprague-Dawley rats were obtained from Simonson
Laboratories (Gilroy, CA) and maintained on normal rat chow. Virgin and
lactating rats were used at 3-3.5 mo of age. Lactating rats,
received ~10 days before the end of gestation, were used on
days 2-5 postpartum. Milk was collected into a clean
sterile syringe upon manual massage of the mammary gland while animals
were under anesthesia with xylazine and ketamine (13 and 87 mg/kg sc,
respectively). Milk release was promoted by injection of oxytocin
(~240 IU/kg ip). Rats were injected intraperitoneally or
intravenously (in the tail vein) with 67Cu or
64Cu(II) as the 1:4 copper-nitrilotriacetate complex
(Cu-NTA), as previously described (19). The
67Cu radioisotope was obtained as a mixture of
64Cu and 67Cu chloride, in HCl, from the
Nuclear Reactor Facility of the University of Missouri, at Columbia;
the 64Cu was 64CuCl2 from the
Mallinckrodt Institute of Radiology Facility of Washington
University, St. Louis, MO (courtesy of Dr. Debra McCarthy). Doses of
radioactive copper injected in 0.6 ml of 0.9% NaCl were 50-300
µCi, containing 20-100 ng of copper. In some cases, rats were
injected intraperitoneally with 125I-labeled rat albumin (5 or 25 µg albumin in 0.9% NaCl, buffered with 10 mM K phosphate) to
monitor its appearance in maternal blood, mammary gland, and milk.
125I-albumin was prepared by treating freshly isolated rat
albumin with 125I-NaI, obtained from Amersham (Amersham
Pharmacia Biotech, Buckinghamshire, UK) by use of Iodogen (Pierce,
Milwaukee, WI) and the protocol from Amersham. Albumin was isolated
from frozen rat plasma by binding to Cibacron Blue (Affigel Blue;
Bio-Rad, Hercules, CA), applied in 20 mM K phosphate buffer, pH 7.1, and eluted with 1.4 M NaCl in the same buffer. Rats were euthanized by
exsanguination, after treatment with heparin and while under anesthesia
with pentobarbital sodium, as previously described (19,
33). Blood plasma was obtained by centrifugation. Whole organs
and tissues were excised. These, as well as portions of milk and
plasma, were directly counted for radioactivity using a Cobra II gamma
counter (Packard Instruments, Downer's Grove, IL). The content of
67Cu or 125I in blood and organs was calculated
as a percentage of the dose. Rat milk and serum samples were stored
frozen at 20°C.
Assays of copper and ceruloplasmin. The total copper content of rat milk was determined by furnace atomic absorption spectrometry, using a Zeeman 800 AA instrument from Varian (Sugarland, TX). Samples of milk were diluted 10-fold with distilled, deionized water before analysis. Standards, made from a 1,005 µg/ml stock solution ("copper atomic absorption standard"; Aldrich Chemical, Milwaukee, WI), were diluted in 1% ultrapure HNO3 (trace metal grade; Fisher Scientific, Pittsburgh, PA). Tissue copper was determined by atomic absorption after wet ashing of 100-mg samples with nitric acid and peroxide, as previously described (59). Ceruloplasmin was assayed by a sandwich ELISA developed with antisera against the 0.6 Rf form of rat plasma ceruloplasmin (33), as previously described (30). The content of 67Cu in milk ceruloplasmin was determined by immunoprecipitation, after removal of the casein, as previously indicated (7), by use of the antibody against the 0.6 Rf form of rat ceruloplasmin raised in a goat (30). Immunoprecipitation of ceruloplasmin from rat plasma was accomplished by the same procedure with and without the addition of protein A beads (Sigma, St. Louis, MO). For immunoblotting, milk and serum ceruloplasmin samples were partially purified by one round of ion exchange chromatography, with DEAE-Sepharose CL-B in 100 mM K phosphate, pH 6.8, and elution with 300 mM of the same buffer, as previously described (33).
Size-exclusion chromatography. Samples (0.5) of plasma were applied to 25-ml columns of Sephadex G150 or Sephacryl S200, and 60 fractions (0.5 ml) were collected for analysis of radioactivity, total protein (absorbance at 280 nm), and, in some cases, ceruloplasmin (by immunoprecipitation).
SDS-PAGE and immunoblotting. Samples of proteins, including immunoprecipitates, were subjected to SDS-PAGE by standard procedures, as previously described (7). Stacking and resolving gels were of 5 and 7.5% acrylamide, respectively. Prestained and unstained high molecular weight standards were from Bio-Rad. Staining was with Coomassie Blue R250. For immunoblotting, samples were transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA) by use of Tobin buffer (25 mM Tris, 192 mM glycine, 1.3 mM SDS, and 15% methanol, pH 8.3) and the Hoefer semi-dry apparatus. The primary antibody was the same as that used for immunoprecipitation; the secondary antibody was rabbit anti-goat IgG conjugated to alkaline phosphatase (Bio-Rad).
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RESULTS |
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Uptake of copper by mammary gland and other tissues during
lactation.
To examine the avidity of mammary tissue for circulating copper,
lactating rats at 2-5 days postpartum were injected
intraperitoneally with 20- to 100-ng doses of 67Cu(II) as
the Cu-NTA complex (see MATERIALS AND METHODS) and killed at various times thereafter. Figure
1, A and
B, shows the total radioactivity (percent dose) absorbed by
mammary gland, liver, and kidney over time. The copper tracer was
rapidly absorbed by the tissues, and maximum uptake was achieved by
1 h. Unexpectedly, most of the copper administered was absorbed by
the mammary gland. More than twice as much tracer entered this tissue
than entered the liver, and uptake by the kidney was seven times less
than into mammary tissue. This contrasts sharply with what normally occurs in virgin (38, 56) and even pregnant rats
(19), where almost all of the incoming copper initially
enters the liver and kidney. As expected, absorption of the tracer by
other tissues of the lactating rats was much lower (Fig.
1C).
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Copper uptake by the mammary gland of virgin rats. Because the mammary gland was so avid for copper during lactation, we wondered whether even in the absence of lactation uptake might be high. Therefore, parallel uptake studies were carried out in virgin rats of the same age. Also, to ensure that the route of isotope administration by injection through the abdominal wall was not causing contamination of the mammary tissue, we decided to compare uptake after giving the 67Cu by tail vein vs. intraperitoneal injection.
Table 1 shows the body and organ weights of the lactating and virgin rats examined. The lactating rats were slightly heavier and had a 15% greater liver weight, as might be expected. The weight of the mammary gland was sixfold greater in lactation, making it comparable in size to the liver. This in itself would be expected to increase the rate of copper uptake by the mammary gland.
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Ceruloplasmin in the milk. The ceruloplasmin and copper contents of rat milk were also assayed, as was the time course of appearance of tracer copper in milk ceruloplasmin. The copper concentration of the rat milk at 2-5 days postpartum averaged 3.3 ± 0.6 (SD) mg/l (n = 8), and the ceruloplasmin concentration (measured by ELISA) was 10 ± 3 mg/l (n = 7). The proportion of radioactive copper in the milk associated with ceruloplasmin at various times after administration of the isotope was also measured after precipitation with specific antibody. The proportion of total tracer with ceruloplasmin was identical at all time points examined (1-122 h) and averaged 27 ± 8% SD; n = 24. This indicates that copper entering the mammary gland went as rapidly into ceruloplasmin as it did into other forms of copper entering the milk. It also proves that the ceruloplasmin in the maternal blood circulation could not have been the primary source of the ceruloplasmin in the milk: by the time maximal labeling of milk ceruloplasmin had occurred, little or no ceruloplasmin labeled with 67Cu had appeared in the maternal blood circulation (see Uptake of copper by mammary gland and other tissues during lactation).
That the copper released into the milk and milk ceruloplasmin was absorbed by the suckling rat pups was verified, as indicated in Table 2. Groups of six pups were suckled for 8 or 24 h on dams that had earlier been injected with 67Cu. (The time of injection vs. start of suckling varied widely.) Clearly, intestinal absorption of copper had occurred, although compared with what was in the gastrointestinal tract, only a small proportion was in liver and spleen of the pups. Radioactivity in other parts of the carcasses was not measured.
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Appearance of albumin in the milk.
To further confirm that most of the ceruloplasmin in the milk was not
coming from the maternal blood plasma, traces of
125I-albumin were administered to lactating rats to label
the maternal plasma pool, and amounts of radioactive albumin in the
milk and plasma 1 h later were recorded. One hour after injection,
there was a large amount of 125I-albumin in the maternal
blood plasma and very little in the milk: 1,226 ± 400 (SD) cpm/ml
(×103) in the plasma (3 rats); 36 ± 30 cpm/ml
(×10
3) in the milk from the same animals (thus 2.9 ± 2.2% of that in plasma). Size-exclusion chromatography of the
plasma samples indicated that 64% of the 125I was with the
albumin at this time (Fig.
5A). The rest (35%) was
associated with a larger protein. In the case of the milk (Fig.
5B), 26% of the radioactivity was with albumin, and the rest was with the larger component (1 h after 125I-albumin
injection). On the basis of radioactivity actually with albumin, the
ratio of 125I in milk to plasma albumin was 0.012. Thus,
1 h after injection, very little 125I-albumin had made
its way from the plasma into the milk.
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DISCUSSION |
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We have demonstrated in the rat that lactation has a marked effect on how copper is distributed to tissues once it has entered the blood. As confirmed here for the normal state, most of the copper entering the blood plasma first travels directly to the liver and kidney (38, 47, 54, 56). In lactation, this changes markedly, 60% of the administered tracer now going directly to the mammary gland. Our studies, the first to follow this process, showed that lactation caused a 20-fold increase in the rate of copper uptake by the mammary gland. This was accompanied by a 6-fold increase in mammary tissue mass, as well as a rise in tissue copper concentrations from 3 to 13 µg/g wet wt. Total copper in the mammary gland thus went from ~8 to ~160 µg, exactly paralleling the 20-fold increase in rate of copper uptake.
Despite the diversion of so much incoming copper to the mammary gland, the concentration of copper in the liver was not diminished by lactation. This is consistent with earlier reports that intestinal copper absorption increases in pregnancy (11, 46) and that biliary copper excretion diminishes (52), only returning to normal in the 3rd wk of lactation, when pups begin to ingest solid food.
Copper that entered the mammary gland went almost immediately into the milk, and ~25% went into milk ceruloplasmin. The time course of radiocopper appearance in this protein exactly paralleled that for the nonceruloplasmin fraction, suggesting a common pathway for entry of the metal into these two milk compartments. The mammary cells concentrated the copper before releasing it to the milk. This was evident from the finding that, 1 h after injection, the concentration of tracer in the milk was 20-fold higher than that of the blood plasma or mammary tissue as a whole, and that of mammary tissue was 4.5 higher than in plasma. The exact mechanisms by which copper entered mammary epithelial cells and found its way into the milk and milk ceruloplasmin remain to be established. However, it is possible that copper entered the cells via a transporter, CTR1, first identified in yeast (62). Export of copper from cells and its insertion into ceruloplasmin within hepatocytes have been linked to the function of two P-type ATPases, ATP7A and ATP7B, defective in Menkes and Wilson diseases, respectively, and found within the trans-Golgi network (TGN) or at the cell surface (33, 40, 60). Copper is delivered to these transporters by the copper "chaperone" ATOX1/HAH1 (4, 17, 42). In contrast to most other cells, the mammary epithelium expresses both of these ATPases (1, 2, 6, 53), so it is unclear whether only one or both are involved in conveying the incoming copper to milk. Ackland et al. (1) found that lactation enhanced expression of ATP7A in the TGN of breast epithelial cells, as well as in the TGN and endosomes of PMC42 breast carcinoma cells treated with lactational hormones (3). However, mutation of the other ATPase, ATP7B, in the "toxic milk" mouse (6, 53) results in a lower copper content of the milk, implying that this ATPase also has a role in milk production.
The ceruloplasmin content of rat milk, determined by the ELISA method here employed, was in the range of what we had previously reported for milk from humans and pigs (7, 59); similarly, our total milk copper values were in the range of those previously reported for rats and mice (6, 7, 50, 52, 59). Our values for the copper content of the nonlactating mammary gland (3 mg/g) appear to be the first ones published for any species. Lower values of 9.7 and 4.0 mg Cu/g (dry wt), respectively, for lactating mammary gland of mice with and without the "toxic milk" mutation (modeling Wilson disease), have just been reported (53), but the day of lactation was not given and might make a big difference (7, 59).
The findings presented here prove conclusively what we had surmised from previous studies, namely, that much of the ceruloplasmin in milk comes from mammary cells and not from the maternal blood plasma. Most of the tracer 67Cu entered the mammary gland, milk, milk ceruloplasmin, and other maternal tissues already within the 1st h after injection. During that period, no 67Cu/64Cu-labeled ceruloplasmin could be detected in the maternal plasma, confirming what had been documented previously for nonlactating rats (19, 56). This is because ceruloplasmin is not part of the exchangeable plasma copper pool. Newly absorbed incoming copper does not bind directly to plasma ceruloplasmin but must first enter the liver and be pumped by ATP7B into the TGN, where it binds to ceruloplasmin newly synthesized on endoplasmic reticulum-bound polyribosomes (34, 60). This must happen before 67Cu/64Cu-ceruloplasmin appears in the blood. Because no tracer-labeled ceruloplasmin was present in the maternal plasma in the 1st h, that in the milk must have come from cells of the mammary gland producing the milk. mRNA for ceruloplasmin is expressed by the milk-producing epithelial cells of the mammary gland (15). Indeed, we have documented in pigs that total amounts of ceruloplasmin mRNA in the mammary gland increase markedly with lactation (7). Consistent with these findings are those of Puchkova et al. (41), who found in rats that radiolabeled ceruloplasmin appeared in the milk much more rapidly than in blood plasma after injection of a 14C-labeled amino acid.
This does not mean that some milk ceruloplasmin did not come from the maternal blood plasma. It is well known that there is paracellular leakage of plasma proteins into the milk (32). We confirmed this by use of 125I-labeled rat plasma albumin, showing that small amounts appeared in the milk within the 1st h after injection into lactating dams. This is consistent with recent findings (48) on the gradual appearance in milk of 125I-labeled recombinant human serum albumin after its injection into rats. At 1 h, we found that the concentration of rat 125I-albumin in milk was about one-eightieth of that in maternal plasma. One would expect Cp to be transferred at a similar or slower rate, because it is larger. If so, and assuming a Cp concentration of 275 µg/ml, one can calculate that ~3.5 µg/ml of the ceruloplasmin in rat milk might have come paracellularly from the plasma, which is one-third of the total detected.
Evidence from the studies reported here is consistent with previous findings, obtained by exon-specific PCR and Northern analysis, indicating that the liver and mammary gland produce the same kind of ceruloplasmin (7). The apparent sizes of the ceruloplasmins detected by immunoblotting in plasma and milk were also identical, as previously reported by us for pigs (7, 24) and humans (31).
In contrast to what we have reported for certain tissues of nonlactating and pregnant rats (19, 54, 56), ceruloplasmin did not appear to be the blood plasma carrier delivering copper to the secretory epithelium of the mammary gland. Instead, as with delivery of copper to liver and kidney, the exchangeable plasma copper pool appeared to be involved. In normal rats, this pool is comprised principally of copper bound to the macroglobulin transcuprein, and that bound to albumin (23, 25, 28). Both of these proteins bind copper very tightly and rapidly exchange it with each other (23, 25, 28). The same two components (transcuprein and albumin) appeared to be the only plasma substituents to carry radiocopper during the 1st h after its administration, as shown by the gel filtration profiles obtained in Sephadex G150 and Sephacryl S200. Moreover, it is worth noting that the proportion of radioactivity binding to the macroglobulin/transcuprein component (eluting in or near the void volume) was larger in the case of the plasma from lactating rats than is observed in rats not in lactation (23, 25, 28, 54, 56). This suggests that transcuprein expression may be upregulated in lactation.
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ACKNOWLEDGEMENTS |
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This work was supported in part by minigrants from California State University, Fullerton, and in part by National Science Foundation (NSF) Grant DUE 9451595. The 64Cu was obtained on the basis of work supported by the National Institutes of Health under Grant R24 CA-86307. Any opinions, findings, and conclusions or recommendations expressed are those of the authors and do not necessarily reflect the views of the NSF.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. C. Linder, Dept. of Chemistry and Biochemistry, California State Univ., Fullerton, CA 92834-6866 (E-mail: mlinder{at}fullerton.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
June 4, 2002;10.1152/ajpendo.00115.2002
Received 14 March 2002; accepted in final form 28 May 2002.
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