ANNALS O F CLINICAL AND LABORATORY SCIEN CE, Vol. 26, No. 3Copyright © 1996, Institu te for C linical Science, Inc.

Early Breast Cancer, Diet, and Plasma Copper Fractions*J. T. DABEK, M.D., Ph.D.t M. HYVONEN-DABEK Ph.D.,* T. KUPILA-RANTALA, M.Sc., Lie. Phil.,* M. HARKONEN, M.D., Ph.D.,t and H. ADLERCREUTZ, M.D., Ph.D.t

tUniversity of Helsinki, Department of Clinical Chemistry,

Helsinki University Central Hospital Helsinki, Finland

andtUniversity of Helsinki, Department of Physics,

Helsinki University, Finland

ABSTRACTIn a study of diet and early breast cancer, blood plasma copper has been

analyzed by Proton Induced X-ray Emission analysis as both total copper (P-Cu) and that ultrafiltrable from plasma (P-edu-Cu) through membranes with a cut-off at molecular mass 10,000 after equilibration with disodium ethylene diamine tetraacetic acid (EDTA) at 4°C. Ceruloplasmin (P-cer) was also measured using nephelometry of anticeruloplasmin monoclonal antibody-ceruloplasmin complexes. Dietary copper intake per day (D-Cu) was assessed over a five-day dietary record period and calculated from dietary components using a computer program. P-edu-Cu correlated sig­nificantly with both D-Cu and ceruloplasmin while P-Cu correlated only with ceruloplasmin. Further, ceruloplasmin did not significantly correlate to D-Cu. Hence, P-edu-Cu better reflects copper status than do P-cer or P-Cu as it relates to both the major copper enzyme in plasma and to daily copper intake. This may be important in drawing conclusions about the significance of copper in disease states where copper fractions other than ceruloplasmin may be most important owing, for example, to oxidative properties. Categorization as cancer or normal, by copper parameters (D-Cu, P-edu-Cu, P-Cu, P-cer), was studied in multiple correlation. In particular, the ratio P-cer/P-Cu and the ratio P-edu-Cu/D-Cu were signifi­cantly related to disease. Irrespective of age (pre- and post-menopausal), highly significant differences between normals and early stage breast can­cer patients were seen with p < 0.0001 to p < 0.01. The precise role played by plasma and dietary copper fractions deserves continued attention in view of the present and earlier results in cancer studies.

* Send reprint requests to: J. T. Dabek, M.D., Ph.D., University of Helsinki, Department of Clinical Chemistry, Helsinki University Central Hospital, Haartmaninkatu 4, FIN-00290 Helsinki, Finland.

2150091-7370/96/0300-0215 $01.80 © Institute for Clinical Science, Inc.

2 1 6 DABEK, HYVONEN-DABEK, KUPILA-RANTALA, HARKONEN, AND ADLERCREUTZ

IntroductionEarlier, the role of copper was consid­

ered, measured by proton induced X-ray emission (PIXE) in early breast cancer patients as compared to pre- and post­menopausal control women on vege­tarian or omnivorous diets. There was an imbalance between total plasma copper and ceru lo p la sm in in the cancer patients.1,2 The study is continued by analyzing for freer copper by PIXE as ultrafiltrable copper passing through membranes with a cut-off at molecular mass 10,000, after addition of ethylene diamine tetraacetic acid (EDTA) and equilibration at 4°C overnight. Proton induced X-ray emission is sufficiently sensitive and precise for this and gives the possibility of simultaneous multi- elemental analysis. The results indi­cate that copper could be behaving dif­ferently in cancer patients than in normal ones. Copper movements could be freer with certain adverse effects of copper, such as oxidation-reduction reactions, more prevalent. Since the patients were in an early stage of breast cancer, this could have implications for the develop­ment of cancer.Materials and MethodsS t u d y D e s ig n

The study was performed as part of a wider study on the relationship between diet, hormones, and breast cancer in women. For both the premenopausal (pre-m) and postmenopausal (post-m) cate­gories, there were two control groups, om nivorous (OMN) and vegetarian (VEG), and a patient group including patients with mainly stage I and a few stage II breast cancer subjects (BCA).Su b j e c t s St u d i e d

The OMN groups included 14 pre­menopausal women (mean [SD] age 33 [6]) and 11 postmenopausal women (age

57 [5]). For the VEG, the pre-m group comprised 12 subjects (age 34 [7]) and the post-m 11 (age 59 [5]). The corre­sp on d in g pre-m and post-m BCA included 13 (age 39 [7]) and 10 (age 66 [6]) women, so that they were, on aver­age, about five years older than their con­trols. The weights of the subjects studied varied from 42 kg to 89 kg, mean 61.5 kg, and were roughly normally distributed. Only the post-m OMN had significantly higher weights, mean (±SD) 69 ± 12 kg, than on average, p < 0.05. The subjects’ heights, mean 163.8 cm, range 151 to 174 cm, were also fairly normally distributed, and analysis of variance showed the pre-m OMN to be slightly taller than the others (167 ± 5 cm, p < 0.05). The body mass index calculated as weight/(height)2 ranged from 17.3 to 34.6 kg/m2, mean 22.9, and was significantly higher in the post-m OMN (mean 26.3, p < 0.01).B l o o d Sa m p l e s

For both patients and controls, blood samples were taken following an over­night fast on at least three consecutive days. The plasma was separated, pooled, and then frozen and stored at -20°C un til a n a ly zed . R ep eat sam p lin g occurred after an interval of several months; typically four samplings were performed over about a year. Thus, a minimum of 12 samples were obtained for each individual (four groups of three), and each measured value represents the mean (by pooling) of three samples col­lected on three consecutive days.D ie t a r y A s s e s s m e n t

The OMN consumed all types of food. The group labelled VEG did not eat red meat, only two ate fish as well as eggs, and the rest were lactovegetarians. They had been vegetarians for a mean of 10.3 years and for at least 1.5 years. Diet was assessed over five days four times a year

EARLY BREAST CANCER, DIET, AND PLASMA COPPER FRACTIONS 2 1 7by weighing dietary components on a let­ter balance and analyzing for nutrient composition by a computer program called Nutrica originally developed at the Finnish National Pensions Institute.M e a s u r e m e n t o f P l a s m a C o p p e r L e v e l s

Plasma copper levels were determined from dried plasma using proton-induced X-ray emission (PIXE) analysis where characteristic X-rays emitted from target atoms under proton bombardment are detected. The absolute concentrations of copper were determined from standard curves prepared by adding increasing volumes of Titrisol standard solutions to replicate samples of pooled normal serum, as described earlier.3 Briefly, plasma or serum samples are dried to constant weight under moderate vacuum and pressed into pellets. There is no chemical treatment, which minimizes contamination and which is a trouble­some source of error in trace element work. Precisely 2.4 MeV protons from the2.5 MV Van de Graaff Accelerator of the University of Helsinki emerging through a 7.5 jxm thick Kapton® (polyimide (C22H10O5N2)x)* exit window impinge on the dried sample. This excites electron transitions, and, hence, produces charac­teristic X-ray emission which is detected by a high resolution solid state germa­nium detector coupled to a microcom­puter used as a multichannel analyzer. The laboratory’s own gravimetrically prepared standards, Seronorm®+ and Seronorm protein®t were used as cali­bration standards and control samples.M e a s u r e m e n t o f P l a s m a C e r u l o p l a s m i n L e v e l s

Ceruloplasmin levels were measured from 50 jjlI plasma diluted 10 times in 450

* duPont, Wilmington, DE.t NyCoMed, Oslo.

|xl of filtered (Millex 0.45 |xl filter)* 4 per­cent polyethylene glycol 6000 (PEG 6000) in physiological saline buffered to pH 7.2 in 70 mmol/1 potassium phosphate buffer (PBS). Precisely 20 |xl of sample were reacted in 1.0 ml of 4 percent PEG- PBS (from which a background scattering reading was taken) with 100 jxl of an antiserum (Behring OUIE 08/09),§ diluted 1:20 with filtered 4 percent PEG- PBS in a 0.5-hour incubation at room temperature. Light scattering at 610 nm was measured using a Transcon 102 FN fluoronephelometer.11 For the calibra­tion of the assay, Behring Institute stan­dard serum (OSAU 06107; 0.203 g/1 ceru­loplasmin) was used; dilutions (from 1:2.5 to 1:30) were prepared in 4 per­cent PEG-PBS. All samples, standards, and reagents were equilibrated to room temperature (24°C).

M e a s u r e m e n t o f P l a s m a U l t r a f i l t r a t e C o p p e r C o n c e n t r a t io n s

To 1900 |xl plasma 100 jxl 0.1 M Na2 EDTA (Na2C10H14N2O8 • 2HaO; Titriplex III)H was added and allowed to stand overnight at 4°C and was shown to be very pure using the PIXE-method. The pH of the ultrafiltrate through mem­branes with a cut-off at molecular mass 10,000** was around 7. Because of the cut-off of the membranes the ultrafiltrate contained no ceruloplasmin total protein or subunits (Mol. mass ca. 19,000) and no larger serum proteins like albumin (Mol. mass. —68,000) or transcuprein (Mol. mass —270,000).

The amount of ultrafiltrate was about 0.5 ml and was sufficient for PIXE anal-

t Millipore, Molsheim, France.§ Behring Werke, Marburg, Germany.11 Elomit, Helsinki, Finland.11 Merck, Darmstadt, Germany.** C e n t r is a r t I , S a r to r iu s AG, G ö t t in ­

gen, Germany.

218 DABEK, HYVONEN-DABEK, KUPILA-RANTALA, HARKONEN, AND ADLERCREUTZ

oQ .ao

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PREMENOPAUSE POSTMENOPAUSEFIGURE 1. Total plasma copper levels in pre- and postmenopausal omnivorous (OMN) and vegetarian

(VEG) controls and breast cancer patients (BCA).

ysis. The ultrafiltrate was pipetted onto thin Kapton®* foils and allowed to dry on an area of about 5 mm diameter. An infra­red lamp was used to give a temperature of 40°C at the Kapton®* membrane. Pre­cisely 200 |xl of sample were applied thus to form a dry target for PIXE. The detec­tion of characteristic X-rays and spectral analyses were performed as for total plasma copper.3

St a t is t ic a l M e t h o d s

Results were analyzed using t-tests, the Wilcoxon U-test, analysis of variance (ANOVA), as well as by linear correlation analysis and Kendall’s rank correlation test. Unless otherwise stated, p-values quoted for the results are for non-para-

* duPont, Wilmington, DE.

metric tests for clarity with regard to pos­sible non-normality. Tabulated and sum­marized data are presented as arithmetic or geometrical means and ranges, includ­ing the range from — 1 to +1 standard deviation of the geometric distributions.

ResultsThe plasma copper concentration

ranged from 11.8 to 29.5 jmiol/1 (figure 1 and table I), with a variation in individu­als of ±11 percent (SD). In the pre­menopausal BCA patients, serum copper was significantly greater than in their controls (p < 0.03); postmenopausally, there was no significant difference between groups.

Ceruloplasmin values ranged from 0.148 to 0.637 g/1 (figure 2 and table I), with a variation in individuals of ±15

TABLE I

EARLY BREAST CANCER, DIET, AND PLASMA COPPER FRACTIONS 2 1 9

Plasma Copper, Ceruloplasmin, and Ratios of Copper-to-Ceruloplasminab

ControlsOmnivores Vegetarians Patients

PremenopauseCopper

(H.mol/1)16.5 ± 0.30 (51) 16.7 ± 0.43 (48) 18.7 ± 0.62° (48)

Ceruloplasmin(g/i)

0.327 ± 0.007 (52) 0.322 ± 0.011 (48) 0.335 ± 0.009 (48)

Ratio(ng/g)

3.20 ± 0.07 (51) 3.40 ± 0.086 (48)

Postmenopause

3.44 ± 0.061 °'d (48)

Copper(|xmol/l)

18.4 ± 0.40® (45) 18.5 ± 0.47® (38) 18.2 ± 0.31 (32)

Ceruloplasmin(g/i)

0.387 ± 0.013f (45) 0.355 ± 0.011'(40) 0.309+ 0.0113 (33)

Ratio

(ng/g)3.02 ± 0.074 (45) 3.22 ± 0.083 (38) 3.94 ± 0.096h'' (32)

a Values are means ± standard error of number of determinations in parentheses. b Statistical significance (by U test) is as follows: significant difference between patients and controls,

(°p < 0.05; 9 p < 0.01;h p < 0.001); significant difference between premenopausal and postmenopausal controls (e p < 0.05;f p < 0.01); significant difference between premenopausal and postmenopausal patients (' p < 0.001); d versus premenopausal omnivores (p < 0.01).

percent (SD). The levels in the pooled premenopausal controls were signifi­cantly lower than those in the pooled postmenopausal controls (p < 0 .001), with no significant effect of diet. The postmenopausal BCA patients had sig­nificantly lower ceruloplasmin levels than the pooled postmenopausal controls (p < 0.001).

The copper-to-ceruloplasmin ratios (figure 3 and table I), ranging from 2.0 to 5.85 |xg/g were significantly higher in the pooled BCA patients than in the pooled controls (p < 0.001). Although the pattern in the pre- and postmenopausal groups was similar, the most striking difference was in the latter (p < 0.001 for BCA vs. omnivores; p < 0.01 for BCA vs. vege­tarians). Premenopausally, the patients also had a higher ratio than the controls;

however, only for the omnivores was the ratio significantly higher in patients than in controls (p < 0.04).

The EDTA-chelatable ultrafiltrable copper (P-edu-Cu) levels in plasma of patients and controls with an omnivorous or vegetarian diet are shown in figure 4. As expected for chelatable ultrafiltrable copper, there is a wide range from 75.2 (xg/l to 516 |xg/l geometric mean 227 (mean +1 SD 324, mean — 1 SD 160). Even after accounting for the logarithmic distribution of the results, there were no significant differences betw een the groups. Unlike total plasma copper and ceruloplasmin, the P-edu-Cu correlated significantly with the dietary copper intake (figure 5 correlation coefficient 0.2, p < 0.005). The correlation, how­ever, weakened for higher dietary cop-

2 2 0 DABEK, HYVONEN-DABEK, KUPILA-RANTALA, HARKONEN, AND ADLERCREUTZ

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PREMENOPAUSE POSTMENOPAUSEFIGURE 2. Plasma ceruloplasmin levels in pre- and postmenopausal omnivorous (OMN) and vegetarian

(VEG) controls and breast cancer patients (BCA).

per intakes (figure 6); after the mid-range of diet, copper P-edu-Cu levels tended to plateau.D ie t a r y C o p p e r a n d T o t a l F ib r e

Dietary copper concentrations calcu­lated from the weighed five-day dietary components by computer program are shown in table II. The values are given for the different pre- and post-m groups as crude data and also normalized per square meter indexed as the square of height. Also shown are the fiber intakes in the different groups. Pre-menopaus- ally, the copper intakes were lower in the patients than in the controls (p < 0.05); post-m, the vegetarians had significantly higher copper and fiber intake than in both other groups.

D i s c r i m i n a t i o n B e t w e e n B r e a s t C a n c e r a n d N o r m a l C o n t r o l s

In this study, because dietary copper, dietary fiber, total P-Cu and EDTA- chelatable ultrafiltrable P-Cu (both expressible as per plasma dry mass or wet volume; the PIXE method involves dry­ing of samples) as well as P-ceruloplas- min were all available, it was possible to use all these variables together in analy­ses. The results showed better discrimi­nation between dietary normals and breast cancer patients than by using plasma variables alone. For the ratio of the plasma variables P-Cu/P-cer (figure 3), the significances of the higher ratios in cancer patients over controls were as follows: for all controls against all patients, ie, overall, p < 0 .001; pre-m

EARLY BREAST CANCER, DIET, AND PLASMA COPPER FRACTIONS 2 2 1

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OMN VEG BCA OMN VEG BCA

PREMENOPAUSE POSTMENOPAUSEFIG URE 3. The ratio of plasma copper to ceruloplasmin (|xg/g) in pre- and postmentopausal omnivorous

(OMN) and vegetarian (VEG) controls and breast cancer patients (BCA).

<DCLCLOO ^V CT>Ì 2 <0 O= E< 4 -CO

~f— ■----1----'— i— 1----1----'— I— '— rOMN VEG BCA OMN VEG BCA

F i g u r e 4 . Ultrafiltra- ble EDTA-plasma copper levels in pre- and post­menopausal omnivorous (OMN) and vegetarian (VEG) controls and breast cancer patients (BCA).

PREMENOPAUSE POSTMENOPAUSE

2 2 2 DABEK, HYVONEN-DABEK, KUPILA-RANTALA, HÀRKONEN, AND ADLERCREUTZ

F i g u r e 5. The sig­nificant positive linear c o r r e la t io n b e tw e e n dietary copper intake and ultrafiltrable copper from EDTA-plasma.

«5OJDuCUa.oU»3«uEal_

6005 0 0 .4003 0 0 .200100

Dietary Copper (mg/day)BCA vs. pre-m OMN, p < 0.001; pre-m BCA vs. pre-m VEG, p < 0.01; post-m BCA vs. post-m OMN, p < 0.04.

Statistically more significant discrimi­nation between breast cancer and con­trols was seen using the compound ratio of edu-Cu/dt-Cu to P-Cu/P-cer (figure 7), where edu-Cu is expressed per unit of plasma dry weight and dt-Cu is normal­ized per kg body weight. In breast can­cer, the ratio is higher than in dietary con­

trols both pre- and postmenopausally. For all controls against all patients, ie, overall, p < 0.0005; pre-m BCA vs. pre-m OMN, p < 0.001; pre-m BCA vs pre-m VEG, p < 0.05; post-m BCA vs post-m OMN, p < 0.03; post-m BCA vs post-m VEG, p < 0.0005.

When the copper tightly incorporated into plasma proteins (ceruloplasmin) was calculated as total P-Cu minus P-edu-Cu (abbreviated b-Cu), a highly significant

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Dietary Copper (mg/day)FIGURE 6. The significant positive second order polynomial correlation between dietary copper intake

and ultrafiltrable copper from EDTA-plasma, indicating the plateauing of the relationship.

TABLE II

Daily Dietary Copper and Total Fiber Intake as Geometric Means with Single Standardized Ranges

EARLY BREAST CANCER, DIET, AND PLASMA COPPER FRACTIONS 2 2 3

ControlsOmnivores Vegetarians Patients

Diet copper 1.71 (1 .40 - 2.09)Premenopause3

1.56 (1 .28 - 1.89) 1.37 (1 .1 0 - 1.70)(mg/day)

Diet copper 0.61 (0 .50 - 0.75) 0.56 (0 .47 - 0.68) 0.51 (0 .4 0 - 0.64)(mg/m2/day)

Diet fiber 18.6 (13.9 -24 .9 ) 23.5 (18.4 -30 .0 ) 13.4 (9.4 -18.9)(g/day)

Diet copper 1.30 (0 .99 - 1.72)Postmenopauseb

1.61 (1 .34 - 1.94) 1.24 (1 .0 5 - 1.47)(mg/day)

Diet copper 0.50 (0 .4 0 - 0.63) 0.61 (0.50 - 0.75) 0.47 (0.41 - 0.53)(mg/m2/day)

Diet fiber 16.6 (12.7 -21 .7 ) 20.2 (13.8 - 29.7) 15.5 (12.5 -19.3)(g/day)

a Premenopausally, the patients’ copper and fiber intakes were significantly less than in the normals (p < 0.05).

b Postmenopausally, the vegetarians' copper and fiber intake were greater than the other groups’ (p < 0.05).

difference between both pre- and post- was seen (figure 8). The function used is:menopausal breast cancer patients and (b-Cu/P-cer) x (edu-Cu/dt-Cu). Here,both vegetarian and omnivorous controls b-Cu = plasma tightly incorporated cop-

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F i g u r e 7 . D iscrim i­nation b e tw een b reast cancer and controls using the plasma ultrafiltrable co p p er/d ie ta ry co pp er and the plasma total cop­per/ceruloplasmin ratios. Discriminator: (edu-Cu/ dt-Cu)/(cer/P-Cu), where: P-edu-Cu = edta-ultra- filte red C u/plasm a dry weight; P-Cu = plasma copper; cer = p lasm a ceruloplasmin; and dt-Cu = dietary copper/weight. T he le v e ls in b re a s t cancer patients are sig­n ifican tly e le v a te d (p < 0 .0 0 0 5 ).

PREMENOPAUSE POSTMENOPAUSE

2 2 4 DABEK, HYVONEN-DABEK, KUPILA-RANTALA, HARKONEN, AND ADLERCREUTZ

F i g u r e 8 . D iscrim i­nation be tw een b reast cancer and controls using the plasma ultrafiltrable copper/dietary copper per sq u a re -m e te r and th e plasm a b ound copper/ ceruloplasmin ratios, tak­ing into account colloid con ten t o f p lasm a and dietary fibre. Discrimina­tor: (b-Cu/cer).(edu-Cu/ dt-Cu), w here: b-Cu = plasma bound copper; ie, total Cu-ED TA -ultrafil- tered Cu; cer = plasma ceruloplasmin; P-edu-Cu = E D T A -u ltra filte red C u/plasm a dry w eight; and dt-Cu = dietary cop­per x d ie t fibre/height squared. The levels in breast cancer patients are significantly elevated (p < 0.0001).

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PREMENOPAUSE POSTMENOPAUSE

per, ie, total P-Cu m inus P-edta- ultrafiltered Cu; P-cer = plasma cerulo­plasmin; edu-Cu = EDTA chelated ultrafiltered Cu/plasma dry weight; dt-Cu = dietary copper x diet fibre/ height squared. For all controls against all patients, ie, overall, p < 0.0001; pre-m BCA vs. pre-m OMN, p < 0.001; pre-m BCA vs. pre-m VEG, p < 0.001; post-m BCA vs. post-m OMN, p < 0.001; post-m BCA vs. post-m VEG, p < 0.0005.

DiscussionBreast cancer causes about 45,000

deaths per year in the USA, and, like other cancers, arises owing to gene defects causing changes in control of cell metabolism. In particular, the control of the cell cycle is defective leading to unregulated growth of the defective, cancerous cells. The diverse defects4 include emergence of oncogenes such as c-myc, int-2 and c-erbB2, — with amplifi­cation of DNA in their regions, and loss of heterozygosity on various chromosomes

- lp,lq,3p,7,llp,13q,17p,17q and 18q.5 Suppressor genes like p53 often have defective products. In familial forms of breast cancer, different genetic changes may be at work, as with the BRCA1 and BRCA2 genes,7 but these syndromes also have a relationship to sporadic breast cancer,8 eg, where a second mutation involving the good allele to an inherited defective suppressor gene leads to cancer development. The frequency of genetic changes may be correlated with histo­logical tumour grade.6

Hence, any factor which can damage the cell’s DNA (also that of repair genes)9 is potentially important for the develop­ment of cancer and its progression. One category of substances which can induce mutations is metal ions, like CuI/II and Fell/III. These can take part in and cata­lyse oxidation-reduction reactions of the Fenton or Haber-Weiss types yielding hydroxyl radical (*OH) from hydrogen p ero x id e or su p e r o x id e . 10,11 T he hydroxyl radical is highly reactive and destructive to macromolecules and may lead to local damage to the molecules of

EARLY BREAST CANCER, DIET, AND PLASMA COPPER FRACTIONS 2 2 5

life, if its production is uncontrolled. Oxidation may also be due to other active oxygen species, like superoxide radicals and peroxide12 in the absence of sufficient superoxide dismutase13 or cat- alase activities, respectively. Hence, DNA bases can be hydroxylated to yield 8-hydroxyguanine and cytosine or thy­mine glycols. This particularly causes cytosine-to-thymine mismatching during copying, with mutations occurring clus- terwise.14 Copper ions bind to bases and not to the sugar-phosphate moiety (as, for example, Mg does) and Cu-2+ then tends to destabilize DNA. Copper’s capacity to cause oxidative damage in the presence of H20 2 has been reported as 50-fold that for iron.15

Although for some time it had been believed that about 95 percent of plasma copper was associated with ceruloplas­min,16 recent studies have shown that as little as 65 percent may be nearer the truth with about 8 percent in a ca. 270,000 molecular mass protein called transcu- prein, 14 percent attached to albumin, and about 5 percent attached to small molecules like aminoacids.17 Plasma and extracellular copper pass into the cell at least partly by mediation of ceruloplas­min receptors18 on the cell surface which are not internalized themselves in the process. Some have reported transcu- prein copper in malignancies and ele­vated total as well as ceruloplasmin cop­per;19 the elevation in Wilson’s disease and Indian childhood cirrhosis is due to an increase in non-ceruloplasmin copper probably released from the copper laden liver.19 The elevated serum copper after a myocardial infarction would be a simi­lar phenomenon.

Ultrafiltrable copper has been studied in gynecological malignancies with the conclusion that because levels are similar in controls and patients, copper is not the etiological element in these cancers.20 In that work, no chelating agent was used so that freer copper was limited to a very

small fraction of the freer copper of our study. Also, the ultrafiltration mem­branes had a cut-off of 30,000 so that smaller fragments (eg, the 19,000 frag­ments) of ceruloplasm in could have passed through.

The damage inflicted by copper on macromolecules is not necessarily all due to freer copper. Although it has been thought that ceruloplasmin is primarily antioxidant in nature, it can also cause oxidative damage to macromolecules such as low density lipoprotein.21 Again, simple oxidative mechanisms may not account for all the damaging potential of copper in the case of DNA.15 Some seem to have shown in certain systems using spin traps that iron, rather than copper, may be responsible for DNA damage via hydroxyl radicals.11 Even if copper does not produce these in the vicinity of DNA, it could act detrim entally in another way.14

Copper/zinc ratios, which tend to be more statistically significant in advanced cancer, have earlier been used to dis­criminate cancer patients from normals when copper levels or zinc levels alone have not shown this capability. Distin­guishing between cancer and normals in early cancer is more demanding, and pro­vision of efficient prognostic indices yet again more difficult. The patients in this study had early stage breast cancer with, however, statistically significant alterations in the P-Cu/P-cer ratio. The inclusion, in discrimination between normals and cancer patients, of other cop­per parameters like dietary copper and dietary fiber, which can bind metal ions and, hence, influence bioavaila­bility, gave better statistical significances than those for the simple P-Cu/P-cer ratio. Further, the patterns of differences between normals and cancer patients were similar pre- and post-menopaus- ally. Further research involving this more comprehensive approach could help uncover possible important differ­

2 2 6 DABEK, HYVONEN-DABEK, KUPILA-RANTALA, HARKONEN, AND ADLERCREUTZ

ences in copper metabolism between normals on various diets and early can­cer patients.Acknowledgments

Thanks are extended to laboratory technicians Inga Wiik and Helena Taskinen for much help in sam ple prepara tion p rocedures and to Raimo Ingren, B.Sc., for invaluable assistance in the PIXE runs. A grant from the Magnus Ehrnrooth Founda­tion was received by TK-R and the Meltola Hospital is thanked by JD for support and access to comput­ing facilities.

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Adlercreutz H. Evidence for increased non­ceruloplasm in copper in early-stage human breast cancer serum. Nutr Cancer 1992;17:195- 201 .

2. Dabek J, Hyvonen-Dabek M, Adlercreutz H, Harkonen M, Hamalainen E, Ollus A, Kupila- R antala T. S im ultaneous investigation of dietary and plasma copper, zinc, iron and sele­nium in pre- and post-menopausal omnivores, vegetarians and patients with early breast can­cer. J Nutr Med 1994;4:403-14.

3. H yvonen-D abek ML, N ikkinen-V ilkki P, Dabek JT. Selenium and other elem ents in maternal and umbilical serum, as determined simultaneously by proton-induced X-ray emis­sion. Clin Chem 1984;30:529-33.

4. Van de Vijver MJ, Nusse R. The molecular biol­ogy of breast cancer. Biochim Biophys Acta 1991;1072:33-50.

5. Callahan R, Cropp CS, Merlo GR, Liscia DS, Cappa AP, Lidereau R. Somatic mutations and breast cancer. A status report. Cancer 1992;69(6 Suppl): 1582-8.

6 . Tsuda H, Fukutomi T, Hirohashi S. Pattern of gene alterations in intraductal breast neo­plasms associated with histological type and grade. Clin Cancer Res 1995;1:261-7.

7. Wooster R, Neuhausen SL, Mangion J, Quirk Y, Ford D, Collins N, Nguyen K, Seal S, Tran T, Averill D, et al. Localization of a breast cancer

susceptibility gene, BRCA2, to chromosome 13ql2-13. Science 1994;265:2088-90.

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