Neurochemical Research, Vol. 9, No. I1, 1984

COPPER DISTRIBUTION IN THE NORMAL H U M A N BRAIN

E R N E S T O B O N I L L A , E N R I Q U E SALAZAR, JOSE JOAQUIN

V I L L A S M I L , R U D D Y VILLALOBOS, M A G A L Y G O N Z A L E Z , AND

JOSE O M A R D A V I L A lnstituto de Investigaciones Cllnicas

Universidad del Zulia and INBIOMED-FUNDACITE Apartado 1151

Muracaibo, Venezuela

Accepted June 6, 1984

Copper concentration was determined in samples from 38 areas of 7 normal human brains. The grey matter contained higher concentrations of copper than the white matter. Identical areas of the grey and white matter of the cerebral cortex showed significant differences between individuals. In the caudate nucleus the highest concentrations of copper were found in the tail followed by the body and the head, respectively. A negative linear regression between age and brain copper levels was demonstrated.

INTRODUCTION

As a component of numerous metalloenzymes copper plays an important role in the normal functioning of mammalian tissues. Copper is absorbed from the stomach and all portions of the small intestine. Thereafter, it becomes loosely bound to serum albumin and aminoacids and it is widely distributed to the tissues. The copper in ceruloplasmin is not so readily available for exchange. The liver is the main storage organ of the body for copper and it also provides the major pathway of its excretion via the bile (1).

Copper deficiency affects the central nervous system mainly during infancy (2). In fact, Menke's kinky hair syndrome is due to a genetically determined defect in copper absorption from the intestinal mucosa to the blood and is characterized, among other symptoms, by progressive mental

1543 0364-3190/84/110'0-1543503.50/0 �9 1984 Plenum Publishing Corporation

1 5 4 4 BONILLA ET AL.

deterioration (3). On the other hand, Wilson's disease is an inborn error of metabolism that affects copper homeostasis. The genetic abnormality leads to excessive copper accumulation in the liver, brain, kidney, and cornea (1). Other nervous system diseases caused by a dietary copper deficiency have been reported in lambs from copper deficient ewes, goats, pigs, guinea pigs, and rats (1).

In order to get a better insight about the pathophysiology of the nervous system alterations produced by copper deficiency in humans, it is nec- essary to know the regional distribution of this metal in the brain. In two previous studies (4, 5) the determination of the concentration of copper have been limited to only a few brain regions, while Warren et al (6) have studied 25 areas in 9 human brains using a colorimetric method.

In the present work we include results from 38 areas of 7 normal human brains. The copper concentration was determined using a more sensitive flameless atomic absorption spectrophotometric technique.

EXPERIMENTAL PROCEDURE

Methods. Samples weighing between 5 and 55 mg were taken from unfixed brains in which macroscopic examination showed no signs of disease. The autopsies took place from 2 to 4 hours after death. The duramater and the brain samples were cut with scrupulously clean metal-free stainless steel knife, forceps, and scissors. The samples were put into previously washed copper-free glass vials. External contamination with copper was avoided by par- ticular care in obtaining the samples and by rinsing them with doubly distilled demineralized water to greatly reduce the error from blood contamination. The serum copper concentration in our male population was found to be a relatively low 0.65 - 0.28 ixg/ml (mean _+ SD) (7). In the present work because very small tissue samples were dissected from brain the risks of contamination with blood decreased considerably. Joyce (8) has found less than 0.001 ml of blood per gram of brain tissue in killed rats.

The copper content was determined by flameless atomic absorption spectrophotometry using a Perkin-Elmer model 370 AAS with an HGA-2100 graphite furnace. All samples were analyzed by the method of standard additions. The procedures used for glassware cleaning and tissue preparation have been previously described (9). Hollow cathode current, slit setting, and the operating conditions for the graphite furnace were set as suggested by the manufacturer.

Brain regions of seven males ranging in age from 11 to 75 years were analyzed. Two of them (20 and 66 years old) died from multiple traumata without brain lesions. Three (58, 64, and 75 years old) died as a consequence of myocardial infarctions. Among the other two, one (17 years old) died by haemorrhage after a gun shot and the other (11 years old) by drowning. None of the 7 persons had a previous disorder of the nervous system or any other apparent illness different from the cause of death.

Statistical Analysis. The general linear model from S.A.S. (Statistical Analysis Systems) was used for the analysis of variance and the comparison between the means (10). With this procedure we analyzed the following variables: a) amount of copper of all the areas studied in the 7 brains, including gray and white matter; b) concentration of copper in the gray and white matter of the frontal, parietal, occipital, and temporal lobes; c) amount of copper in

COPPER IN THE BRAIN 1545

the head, body, and tail of caudate nucleus; d) correlations between age and brain copper content from all regions.

RESULTS

Copper was found to be unevenly distributed in the human brain (Table I). The gray matter yielded higher concentrations of copper than the white matter (16.73 +_ 0.89 Ixg/g dry weight for gray matter; 10.74 + 2.07 txg/g for white matter). The difference was statistically significant (Table II). The copper concentration ratio of cerebral gray matter to white matter was 1.56. Identical areas of the gray and white matter of the cerebral cortex showed significant difference between individuals (Table II).

In the caudate nucleus, the highest concentration was found in the tail, followed by the body and the head, respectively of the same region. The difference was statistically significant as shown by the analysis of variance (Table III).

No difference was observed for the concentration of copper in the gray matter of the frontal lobe (18.80 _+ 1.15 ~g/g) when compared with the occipital (22.34 + 1.61 txg/g), parietal (17.84 _ 1.59 Ixg/g) and temporal (16.30 _+ 2.32) lobes, but when comparing the content of copper in the gray matter of the occipital lobes with that of the parietal and temporal lobes the difference was significant at the 5% level.

A lineal regression analysis was done to study the functional relation- ship between copper concentration from all regions and age. It showed the following model: Y = 19.66605 - 0.08472X, where Y is the copper content and X, the age. Considering this model it was found a negative lineal regression between age and brain copper concentrations. With in- creasing age a decrease in brain copper content was observed.

DISCUSSION

Warren et al. (6) using a colorimetric procedure found that the copper content of gray matter was two to three times higher than that of white matter. The substantia nigra and locus ceruleus were shown to be par- ticularly rich in copper. They reported concentrations in gray matter as low as 16 txg Cu/g dry weight in thalamus and as high as 201.I ~g Cu/g dry weight in locus ceruleus.

After assaying 10 brain regions by means of atomic absorption spec- trophotometry, Harrison et al. (4) found the highest concentration of cop- per in putamen, globus pallidus and caudate nucleus as well as in frontal

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COPPER IN THE BRAIN 1547

TABLE II ANALYSIS OF VARIANCE FOR THE CONCENTRTION OF COPPER IN ALL AREAS

INCLUDING GRAY AND WHITE MATTER

Source of Sum of Mean Variation DF Squares Square F Probability

Brains 6 1684.32 280.7 6.38 P < 0.01 Matter (gray and

white) 1 1963.08 1963.1 44.59 P < 0.01 Brains. Matter 6 93.97 15.7 0.36 Error 207 9112.42 44.0

TOTAL 220 12853.79

cor tex and cerebel lar cortex. The range of concentrat ions in gray mat ter varied f rom 21 Ixg Cu/g dry weight in thalamus to 44 Ixg/g in putamen.

In 1974 Smeyer s -Verbeke et al. (5) studied 11 regions of 13 brains. They found the highest concentra t ions in gray matter . They also repor ted dif- ferences in the distribution of copper in the gray mat ter of several nuclei as well as differences for the values found in the white matter be tween brains and be tween different areas of the same brain.

In the present study we have analyzed a larger number of brain regions. Again the highest concentra t ions of copper were those of the regions rich in gray mat ter , al though the values we found in gray mat ter were lower than those repor ted previously. For example , Smeyers -Verbeke et al. (5) repor ted 26.1 Ixg Cu/g dry weight for gray mat ter and 12.4 Ixg Cu/g for white matter . Warren et al. (6) found 29.1 Ixg Cu/g for gray and 11.4 Ixg/g for white matter . Our data for white mat ter correspond reasonably well to the values found in the latter two studies.

TABLE III ANALYSIS OF VARIANCE FFOR THE CONCENTRATION OF COPPER IN THREE REGIONS OF

CAUDATE NUCLEUS (HEAD, BODY, AND TAIL)

Source of Sum of Mean Variation DF Squares Square F Probability

Brains 6 769.55 128.26 7.59 P (<0.01) Regions 2 200.31 100.16 5.93 P (<0.05) Error 8 135.17 16.90

TOTAL 16 1105.03

1548 BONILLA ET AL.

As with manganese the highest levels of copper were found in the tail of the caudate nucleus as compared with the body and head of the same structure (11). The reasons for the differential distribution of copper in caudate nucleus is unknown.

The finding of a decrease in the copper concentration of brain with increasing age has to be taken into consideration when evaluating changes in this parameter produced by any disease state. Kishi et al. (12) have reported that copper concentrations in medulla from suckling rats were higher than those in the same region from adult rats.

ACKNOWLEDGMENTS

This work was partially supported by grants from FUNDACION POLAR and FUN- DACITE-ZULIA.

REFERENCES

1. UNDERWOOD, E. J. 1977. Trace elements in human and animal nutrition. Pages 56-108. Academic Press, New York.

2. O'DELL, B. L., HARDWICK, B. C., REYNOLDS, G., and SAVAGE, J. E. 1961. Connective tissue defect resulting from copper deficiency. Proc. Soc. Exper. Biol. Med. 108:402- 405.

3. DANKS, D. M. CAMPBELL, P. E. WALKER-SMITH, J., STEVENS, B. J., GILLESPIE, J. M., and BLOOMFIELD, J. 1972. Menke's Kinky hair syndrome. Lancet 1:1100-1102.

4. HARRISON, W. W., NETSKY, M. G., and BROWN, M. E. D. 1968. Trace elements in human brain: copper, zinc, iron, and magnesium. Clin. Chem. Acta 21:55-60.

5. SMEYERS-VERBEKE, J., DEFRISE-GusSENHOVEN, E., EBINGER, G., LOWENTHAL, A., and MASSART, D. L. 1974. Distribution of copper and zinc in human brain tissue. Clin. Chem. Acta 51:309-314.

6. WARREN, P. J., EARL, C. J., and THOMPSON, R. H. 1960. The distribution of copper in human brain. Brain 83:709-717.

7. PAz-MoNCADA, N., VILLASMIL, J. J., and BONILLA, E. 1981. Distribuci6n de cobre srrico en una poblaci6n suburbana de Maracaibo (Venezuela). Invest. Clin. 22:83-94.

8. JOYCE, D. 1962. Changes in 5-hydroxytryptamine content of rat, rabbit, and human brain after death. Brit. J. Pharmacol. 18:370-380.

9. BONILLA, E. 1978. Flameless atomic absorption spectrophotometric determination of manganese in rat brain and other tissues. Clin. Chem. 24:471-474.

10. STATISTICAL ANALYSIS SYSTEM. 1979. SAS Users guide. Pages 237-263. SAS Institute Inc. Raleigh. North Carolina.

11. BONILLA, E., SALAZAR~ E., VILLASMIL, J. J., and VILLALOBOS, R. 1982. The regional distribution of manganese in the normal human brain. Neurochem. Res. 7:221-227.

12. Ktsm, R. IKEDA, T., MIYAKE, H., UCHINO, E., TSUZUKI, T., and [NOUE, K. 1982. Regional distribution of lead, zinc, iron and copper in suckling and adult rat brains. Brain Res. 251:180-182.