Heavy Metals in the Environment: A threat to Public Health?

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Heavey Metals in the Environment. A threat to Public Health?

Delft Progr. Rep., 12 (1988) pp. 125-191 Received: June 1983

Accepted: April 1983

Papers presented to the

Symposium 'Heavy Metals in the Environment; a Threat for Health?' dedicated to Prof.ir. J.P.W. Houtman

on the occasion of his retirement from IRI and the Delft University of Technology, June 21, 1983

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DELFT PROGRESS REPORT (1988) 12

The Metallobiochemistry of Zinc and Cadmium

B.L. Vallee

Center for Biochemical and Biophysical Sciences and Medicine Boston, Harvard Medical School, Boston, Massachusetts, U.S.A.

131

The honour and opportunity of being invited to address you on the occasion of Professor Houtman's retirement is very much appreciated. However, having met him yesterday, I am convinced that I am not here to help celebrate his retirement as much as to initiate his new career.

Just like careers, the role of metals in biology can be viewed from mul-tiple points of view. In the past, metals have variously been thought to be beneficial, toxic or to serve no purpose at all in biology. It is not very long ago that documentation for the validity of all th ree perceptions seemed to coexist, and that they had equally vigorous advocates; but by now the hypothesis that they serve no biological purpose has faded into oblivion. This dilemma owed much to the fact that many metals are present in 'trace' amounts. I want to address myself today to the general principles underlying the evolution of metallobiochemistry and of its subdisciplines which did not exist a generation ago and give perspective to metals as toxic, environmental agents, a subject which has been of special interest to Prof. Houtman.

The word 'trace' implies something that can be measured qualitatively but not quantitatively, and until about a generation ago that was true for many metals in biology. Therefore it was a nearly mystical subject explored of ten by individuals who had ideas about the problem but who performed few experi-ments to verify or reject them. This has changed. Measurement of very low concentrations of metals in biology has become quite routine. Technical problems have been eliminated to the point where the term 'trace' has become meaningless. That is fortunate, indeed, because to many 'trace' also meant

'unimportant' or 'insignificant' .

Attention to the detection limits of analytical methods for metals has become almost irrelevant now. Yet, the fact that the II-B metals and those of the first transition series all occur in biological matter in similarly low concentrations led to the tacit inference that all of them would be found to perform similar functions. Such an emphasis on similarities of concentrations completely ignored the chemical properties of metals which are unique for each. Not too surprisingly, their biological roles have turned out to be as characteristic as their chemistries, enhanced by co-ordination of metals with

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132 12 (1988) DELFT PROGRESS REPORT a large variety of biological ligands. Such distinctive biological roles of metals are now well appreciated, methods for their detection are standard-ized, and approaches to the study of their functions have merged with those conventional in all other biochemical work. In short, metallobiochemistry is now established as an important subdivision of general biochemistry.

Parenthetically, one might compare the history of the emergent knowledge on metals in biology to that of the function of vitamins. As a class, those also shared low concentrations, but the functions of different vitamins are known to be quite distinct, of course.

Actually, the concentrations in which different metals occur in diverse biological systems prove to vary widely once they can be measured quantitatively. Fig. 1 schematically shows the concentrations of elements in the human body relative to zinco

c

N

0 ₁₀

2 r

w

>

~

W

~

w

_IÖ

2 U

z

~

0 Z

~

m

~

Fig. 1. Body content of metals, relative to zinc

(3-4

g in a 'standard' 70 kg adult).

Calcium, magnesium, sodium and potassium are present in large amounts, fol-lowed by iron, zinc, copper, manganese, molybdenum, cobalt, chromium, vanadium and cadmium.

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f e s e e e e d n I' L-DELFT PROGRESS REPORT (1988) 12 133

It is now apparent that all of those shown do have some biological function, information that was largely unavailable 20 years ago. Table I indicates that in a large part, these advances can be attributed to revolutionary changes in analytical techniques which have taken place since then. When I began to work about 30 years ago about 1 microgram of zinc could hardly be measured and required milligram amounts of sample. It took about one working day to measure zinc chemically in a dozen samples - and then the answer was of ques-tionable validity. Quantitative emission analysis followed, accomplished much the same but did it more effectively on more elements simultaneously.

Table 1. Spectroscopie limits for zinco

Technique

1955

Microchemistry D.e. Arc emission Graphite spark emission 1965

Neutron activation Flame atomie absorption Long tube AA

1975

Microwave plasma emission Flameless atomie absorption

Detection Limit pg 1.0 1.0 1.0 0.1 0.001 0.00002 0.000002 0.00000005 Amount Protein Required mg mg mg mg pg pg ng ng

Neutron activation, flame atomie absorption and long tube absorption spectroscopy came into existence about a decade later; all of them lowered both the detection limits concurrently with the total amount of sample required. Now, microwave plasma emission and flameless atomie absorption spectroscopy measure picogram amounts with a precision of 1-2% on nanogram amounts of sample. Hence, we can now determine picogram amounts of zinc in about thirty samples in one hour with a precision of 1-2%.

As aresuit, the study of the function of metals has taken a different course. In 1912 Gabriel Bertrand stated that the functions of all elements should be considered a continuum. He believed that, potentially, every metal has a biological function which can be assessed against a deficiency state;

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l34 12 (1988) DELFT PROGRESS REPORT

the function under study will increase to a maximum as the metal is

replenished. This maximum physiological response represents phase I of this

dose-response in Fig. 2. 0 _ES I _PH T

T

E F _SE M _BA A 0 X I R I C F N L _{A MA} C IE U_{N TI}

~

I W _N _C _A _C C Co T TI Y Cf) C T L E L Z Y 10 0 0

0

N N GI

a..

S C Cf) W 0:: U (!)

0

...J

0

(IJ

METAL CONCENTRATION

)

Fig. 2. Biological responses to changes in concentration of a metal.

If a given metal does not exist in proper relationship to all other metals

present, essential functions may be hampered causing metal-ion imbalance, the

middle of Fig. 2. Metal concentrations higher than those required to maintain

essential functions may have pharmacologic effects constituting phase 11 of

that figure. At still higher concentrations, every element is potentially

toxic and ultimately lethal. This postulate has turned out to be quite

cor-rect, and the ensuing pattern seems to hold for virtually all elements about

which adequate information exists.

It is therefore inappropriate to consider and classify metals as either

beneficial, toxic or lethal elements; each one can be all of these, dependent

on dosage. Cobalt for instance, present in very small amounts in most

species, becomes incorporated into vitamin B

12 and then becomes essential.

The amount of cobalt in an adult human is of the order of micrograms to

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T _s _s .ls .he .in of .ly >ut mt )st to of -- - -

-DELFT PROGRESS REPORT (1988) 12 135

vitamin B

12 are very high; in fact, it is indispensible. But, if cobalt it-self is administered to human beings much beyond those amounts accounted for by vitamin B12' this will generate an excess of red blood cel Is , i.e. polycythemia; further, when added to beer as an anti-foaming agent, cobalt can cause serious heart disease and death. Analogous but different dosage-dependent effects can be cited for iron, copper and nickel.

The question is, where do metals act? They act in enzyme catalysis, and I shall devote much of my discussion to their function as essential components of enzymes. They are also involved in oxido-reduction, in transport processes across membranes of nerves and muscles, where calcium, magnesium, sodium and potassium perform trigger functions in nerve conduction. These elements move very quickly across boundaries and exchange very fast. Ma~y metals function in subcellular organel les , they stabilize protein and nucleic acid structure, participate in immune phenomena, and zinc, at least, is involved in the transmission of genetic information (TabIe 2).

Table 2. Sites of zinc participation in biologic systems.

Enzymatic catalysis Oxidation-reduction Transport processes

Membrane, nerve, muscle functions Immune phenomena

Function of subcellular organel les Stabilization of protein

Nucleir acid structure

Transmission of genetic information

When metals are an essential part of enzymes, one may think of them in the same manner in which one considers vitamins parts of enzymes, i.e. as co-enzymes. Not too many enzymes were known to contain functionally essential metals until quite recently, mostly because of the analytical problems described above. The discovery that so many metals are functionally indispen-sible components of enzymes has now brought many inorganic chemists and their approaches into enzymology.

Metals interact with certain amino-acid side chains to form specific com-pI exes , functionally constituting an active site (TabIe

3).

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136 12 (1988) DELFT PROGRESS REPORT

Table

3.

Amino-acid side chains which can complex with metals.

-NHCH(R)CO- with as functional group R

group of lysine

(CH2 )3NHC (=NH)NH2

guanido group of arginine

(CH2)n COOH, n = 1,2 carboxyl group of aspartic and glutamic acids

CH2SH, thiol group of cysteine HC=C I I H N::::':C,.-NH H imidazole group of histidine (CH2)2SCH3' thioether of methionine

CH2C,H.OH, phenolic group of tyrosine

CH20H, aliphatic hy-droxyl group of serine and threonine

Iron- and magnesiumporphyrins as weIl as cobalamins are functional, metallo-organic, prosthetic groups of enzymes also.

In a given metallo-enzyme, a number of amino-acid residues, separated from one another in the linear sequence of the protein, are folded in three-dimensiona1 space such as to constitute the binding site for a specific metal, preferentially binding it with a high stability constant. The resul-tant active site complex in effect constitutes a chemical and biological entity (Tabie

4).

The enzyme holds on to the native metal beyond all others, expressing the genetic endowment of this particular apoenzyme. The underlying chemistry is highly specific, but the factors which render it that way are still somewhat mysterious. In certain instances a different metal can be sub-stituted for that present in the native state while activity is maintained, as I shall discuss more specifically for carboxypeptidase.

Toxic manifestations can result either from competition of such another metal when in excess for the specific native-site metal, or, more commonly, from an inactivating interaction of a metal with different amino-acid side chains of an enzyme which are not involved in the active site (Tabie

4).

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- - - - -- ~ ~ - - -

Table

4.

Characteristics of metallo-enzymes and metal enzyme complexes.

Metallo-enzymes

Metallo-enzymes can be characterized chemically, and activity is direct-ly related to metal content.

Metal enzyme complexes

No chemical characterization has been achieved. Only biological activity can 'be employed to evalu-ate an effect of the metal.

For a casual inspection of the field, Table 5 shows metallo-enzymes contain-ing cobalt, copper, iron, manganese, molybdenum, nickel, selenium and zinco

Table 5. Metallo-enzymes.

Metal Number Class (LU.B.)

Co 1 Il Cu 35 I Fe 68 I Mn 6 I, lIl, VI Mo 12 I Ni 8 I, III Se 10 I, Il

Zn 203 I, Il, lIl, IV, V, VI

It is, indeed, a substantial collection by now. Some of these metals entail colour to enzymes containing them, while others are colourless. Thus, copper proteins are blue, iron proteins are red, but zinc, cadmium and magnesium proteins are colourless, all like their respective salts. The colour of iron and copper enzymes constitute 'probe' characteristics and have been important to their recognition as metalloproteins. Hence, much was known long ago regarding the existence of iron and copper proteins and their characteristics. One would not readily be aware of the existence of proteins containing colourless metals unless one employed a method for their detection which does not rely on visible colour. Emission and atomic absorption spectroscopy fit that description, and their development has changed the field. A count in 1983, turned up 203 zinc enzymes in contrast with solely a

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single one 30 years ago, a remarkable advance. In point of fact, zinc is es-sential to the function of a very large number of enzymes. Importantly, each of the six classes recognized by the International Union of Biochemistry oxido-reductases, transferases, hydrolases, lyases, isomerases and ligases -contains at least one zinc enzyme.

How did it get to be this way? The number of zinc enzymes found has in-creased remarkably as a direct function of the now superior state of analytical methods mentioned above together with the increasingly sophisti-cated methods for enzyme isolation. Hardly a month passes by without discovery of yet another zinc enzyme. Since there are such a large number of zinc enzymes, the question now arises: how is zinc partitioned between them? How does a zinc-dependent biological system insure the integrity of the various enzymatic functions that are needed? The chemical structure of ac-tive, enzymatic sites is important in providing an answer to this type of question, and X-ray structure analysis of enzymes has been employed success-fully towards this end. X-ray structure analysis of e.g. carbonic anhydrase has established that its zinc atom is bound by three specific histidines

(Fig.

3).

His 63

His 93

i f

Fig.

3.

Schematic representation of the active site of (human) carbonic

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- - -

In carboxypeptidase the zinc atom is bound to two histidines and one glutamic acid residue, much the same as in thermolysin. In liver alcohol dehydrogenase, composed of two subunits, there are two zinc atoms per sub-unit, i.e. a total of four zinc atoms per molecule. Two atomsper molecule are catalytically active, each bound to two cysteines and one histidine; the other two are inactive, each bound to four cysteines (Fig.

4).

CYS97

Fig.

4.

Schematic representation of the ligands and co-ordination geometry of the catalytic (left) and non-catalytic (right) zinc atoms of equine liver alcohol dehydrogenase.

Thus, some metallo-enzymes employ two or more atoms of the same or of two different elements for different purposes, as indicated in Table

6.

Table

6.

Role of metal atoms in zinc metallo-enzymes.

Examples Catalytic Regulatory Structural Undefined

Carboxypeptidase Zn Aminopeptidase Zn Mg Thermolysin Zn Ca Alcohol Dehydrogenase Zn Zn Alkaline Phosphatase Zn Mg Zn Aspartate Transcarbamylase Zn Superoxide Dismutase Cu Zn

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140

To generalize:

interacts with

12 (1988) DELFT PROGRESS REPORT

a metal atom at the active site of a metallo-enzyme always more than two ligands, i.e. it is at least tri-dentate some-times more, but never less.

Spectroscopie studies corroberate further that the co-ordination

charac-teristics of zinc in metallo-enzymes differ from those in simple model

complexes. In metallo-enzymes, the metal-binding sites are invariably highly

asymmetrie. Their geometry is distorted compared with that of metal complex

ions, either because of unusual bond lengths or odd numbers of ligands. As a

consequence, the spectra of catalytically active metallo-enzymes generally do not resemble those of metal complex ions and may, therefore, be considered

'atypical', as compared with those of convential models. It has been

sug-gested that the active site of metallo-enzymes is 'poised for catalysis', its intended biological function, a situation referred to as the Entatic State. Denaturation concomitantly abolishes both enzymatic function and the spectral features characteristic of the Entatic State.

The state of entasis is thought to constitute an energetically poised

domain and to predict structures of metal co-ordination in enzyme active

cen-ters that deviate significantly from simpler systems currently known. X-ray diffractîon studies of metallo-enzymes have validated the prediction and

ex-istence of such irregular co-ordination geometries. The entatic state

effectively originates in the primary, secondary and tertiary structures of

the apoprotein which dictates the relative spatial position of those residues destined to serve as ligands when it combines with the metal ion. Existent evidence suggests that the metal is not incorporated into the growing, ribosome-bound polypeptide chain, but only into the fully formed protein.

Quite the same it is feasible to exchange the native metal at an active site

and replace it with another which can also at times result in activity. As an

example, removing zinc from carboxypeptidase results in the loss of all its

activity, but substitution with cobalt, manganese, nickel, mercury, lead or

cadmium restores it.

May I point out at this time, that carboxypeptidase is simultaneously a

peptidase and an esterase, and both activities are lost when zinc is removed;

activity is restored on adding zinc back; much the same becomes true when

substituting cobalt, nickel or manganese. The broad substrate specificity of

carboxypeptidase-A is very responsive to the particular metal atom placed at

the active site. Thus, toward peptides the cobalt substituted enzyme is twice

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- - -

enzymes towards esters is nearly the same. The nickel- and manganese-substituted enzymes also hydrolyze both esters and peptides, but the cadmium, mercury, and lead enzymes are significantly active only toward esters, and the Cu(II) enzymes are completely inactive (Tabie

7).

The alkaline-earth derivatives and the enzymes substituted with the remainder of the transition metal ions, are also completely inactive.

Table

7.

Relative activities of metallocarboxypeptidases.

Metal % Peptidase % Esterase

Apoenzyme 0 0 Zinc 100 100 Cobalt II 200 110 Nickel 50 50 Manganese 30 160 Cadmium 5 140 Mercury 0 100 Lead 0 60 Copper II 0 0

Now when cobalt is at the active site, the resultant absorption spectrum be-comes very useful for the study of the entatic state, the mechanism of action, and the understanding of catalytic phenomena in general. It has now become possible to inspect both the characteristics of the cobalt enzyme com-plex as weIl as the activity in a period of milliseconds during catalysis; catalytic intermediates can be identified within 250 nanoseconds. Hence, structural events can be described at the very moment that catalysis occurs at the metal atom. Thus, the presence of a metal can visualize a number of functional and structural features in a manner which has not as yet been ex-ploited in toxicology to the ex tent that it might be possible.

Responding to Professor Houtman's interest in cadmium and zinc, it seems appropriate to mention metallothionein, a favorite of mine which has received relatively little attention by enzymologists. Some 30 years ago, I thought it remarkable that cadmium had never been found to be a native component of a protein, much as its chemistry is so similar to that of zinc th at it is al-ways found in conjunction with that element in geological material. In fact, it had proven difficult to differentiate between these two elements chemi-cally, though their emission spectroscopic characteristics are distinctive,

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142 12 (1988) DELFT PROGRESS REPORT of course. In the early fifties I came across an abstract in the Russian

literature which described relatively large amounts of cadmium in the human

kidney, a finding which I re-examined and verified byemission spectroscopy. Cadmium, in fact, is present not only in human but in the kidneys of many

other species; it is found mostly in the cortex, much less in the medulla.

Cadmium is particularly abundant in the kidney cortex of the horse (TabIe

8)

from which we isolated in 1958 the first cadmium protein now known as met-allothionein.

Table

8.

Cadmium in mammalian kidney (pg/g wet weight).

Species Cortex Medulla

Horse 94 1.7

Human 36 12

CattIe 0.6 0.2

Lamb 3.1 0.6

Dog 0.4 0.1

In addition to cadmium, the protein contained zinc and some copper and iron; ultimately, in human metallothionein we also found some mercury.

Metallothionein is cysteine-rich and of low molecular weight. lts metal content is exceptionally high (up to 12% w/w). Despite 20 years of intensive investigation, the biological function of this protein remains unknown, much as there are abundant numbers of speculative hypotheses. Administration of zinc and other metal ions, including cadmium, copper, mercury and silver can induce metallothionein synthesis. Metallothionein has been identified in the liver, kidney and other organs of a wide variety of mammalian species; similar proteins have been isolated from crustaceans, molluscs and several micro-organisms. All mammalian tissues and the crustaceans examined so far contain at least two distinct forms of metallothionein, of ten denoted 1 and 2. The total metal content of mammalian metallothioneins is constant at 7 g-atom of metal per mole of protein, but crab metallothionein binds only 6 g-atom of metal per mole of protein. The metal composition of the native protein is a function both of the organ from which is it isolated and of the prior treatment of the subject with metal ions. The difference in the metal content of metallothioneins from liver and kidney, apparently independent of

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~ - - - - -- - -

treatment, is especially noteworthy. Thus, the relative abundance of cadmium and zinc in the protein from the kidney is of ten comparable, while zinc is the principal metal in the otherwise identical protein from liver.

The molecular weights of mammalian metallothioneins range from

6,500

to

7,000

,

depending on their metal content. It contains

30-34

residue

%

of cys-teine and the corresponding amount of sulfur exceeds even that of wool. Metallothionein also contains a relatively large proportion of serine, threonine, and basic amine acids. The complete absence of the aromatic amino-acids tyrosine, tryptophan and phenylalanine and of histidine is typical. The amino-acid sequences of metallothioneins from a variety of species have been reported, and the polypeptide chain of all mammalian metallothioneins contain

60

to

61

residues. Cysteines are thought to be the primary metal-binding sites of the protein (Fig.

5),

but it is now agreed that there are additional ligands. Although metallothionein was first recognized because of its cadmium content, from the very beginning major concentrations of zinc and lesser amounts of copper and iron were detected in the native horse kidney protein, suggesting that these elements may replace one another in vivo. In human ren al metallothionein mercury has also been found, perhaps owing to their use of mercurial diuretics. Metal exchange in vitro results in a number of metal-lothioneins containing other metal atoms. The native protein contains neither disulfides nor free thiol groups.

Ever since the discovery of this protein, numerous experiments have been performed to identify its biological role, but none of them have been conclusive. The possibility that the apoprotein, thionein, rather than metal-lothionein, is the active protein and that zinc and other metal ions inhibit

I

s

s" /

"-/

, , / Cd- S,

Cd_ S

I /

---r-Cd -

S

s,

I

\

S

. / ' " / , S ,

_Cd

/

\

"..-S

S ...

/

I

's

S

/

' " /

"Cd--

S

,

Cd_

_S

I /

~Cd-S

S

/

I

\

/

s,

Fig.

5.

Postulated structures of the metal-thiolate clusters in rabbit liver metallothionein based on the 11'Cd-NMR spectrum.

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144 12 (1988) DELFT PROGRESS REPORT it has not been eliminated. Thionein could, for example, function in the me-tabolism of the sulfur-containing amino acids or as an amino acid carrier. Metallothionein might function in some phases of biochemistry which are yet to be explored, e.g. expression of the genetic message, embryonic development or cellular differentiation.

How does metallobiochemistry assist in the evaluation of metals such as cadmium as toxic agents? The current scientific problem is, not so much to establish whether or not a metal has toxic properties, but where toxicity may be expected on a plot such as proposed by Bertrand (Fig. 2). Unfortunately, much of what is known about any given metal refers primarily to a limited portion of the biphasic of that figure, a consideration which is of the greatest importance in efforts to assess risks of metal toxicity.

Metal toxicity differs from other forms of environmental contamination in ways which require both enumeration ffi1d special consideration: 1) while met-als occur in nature, human activities alter their distribution; 2) a determinatioR of the risks of pollution requires quantitative data, but these are of ten limited owing to the inadequacy of methods;

3)

the normal biochemi-cal and biologibiochemi-cal functions of many metals must be differentiated from the effects of their excess on living systems; 4) normal function must be sought and detailed as background for the evaluation of symptoms and sign due to deficiency, intoxication, or imbalance;

5)

finally, in many instances, known true risks due to metals must be distinguished from speculations which are in fashion in order to focus on and address realistically the problems toward which scientists should direct their attention.

In contrast to many other recognized toxic contaminants, metals are normal and intrinsic to earth, water and air in widely different concentrations and geographic areas. Metals have always provided fertile subjects for conjecture and speculation, and their deficiency, intoxication or imbalance has at one time or another been invoked as cause for nearly any human disease for which no specific cause is known. Hypertension, artherosclerosis, sarcoidosis,

lupus erythematosis, rheumatoid arthritis, carcinoma, diabetes, periarteritis nodosa, porphyria, brucellosis, mental retardation, nephritis, senescence and encephalitis have all been suggested to be metal toxicity related. Many of these same disorders whose cause remains unknown have been invoked again recently as potential threats much as there is no evidence for su eh conclu-sions, in part because there have been difficulties in obtaining hard data,

with the lack of precise measurements constituting one major handicap. Current techniques still remain inadequate for accurate determination of some

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-

- -- - - -- - -

metals e.g. cadmium and chromium, that occur only in extremely low concentra-tions in biological material. Thus, determination of the low, 'natural' concentrations of cadmium existent in soil, water or air remain significant analytical challenges.

Metal deficiency in animals causes disease entities th at reflect altered biochemistry (Table

9).

Table

9.

Examples of diseases due to metal deficiencies.

Metal deficiency Iron

Copper Zinc

Cobalt (as vit. B_{1Z )} Manganese Chromium Selenium Inadequate in disease deficiency tain to be supplies both in diseases essential of iron, man and in of ani mals in man. Disease Anaemia

Aortic rupture (cattle) Anaemia; 'Swayback' (sheep) Dwarfism; Gonadal failure; Dermatitis

Anaemia

Gonadal dysfunction; Skeletal abnormalities Abnormal glucose metabolism Liver necrosis;

White muscle disease (lambs)

copper, zinc or cobalt (as vitamin lower animals. Manganese, chromium

B

12) result

or selenium are known, but these elements are not yet cer-When in excess these same metals (and others) are toxic. Thus, Table 10 shows some human diseases resulting from excess iron, copper, zinc or cobalt all of them are also biologically essential metals - and the cardinal manifestations of disease due to intoxication with manganese, selenium, lead, cadmium or mercury, thus far not certain to have essential roles in humans.

The imbalance of metal ions mayalso result in biological malfunction. Indeed, metal-ion antagonism, or the competition of one metal with another for a critical biological site is perhaps the commonest source of pathology which is also more difficult to establish than either metal deficiency or intoxication. Metal-ion imbalance is widespread throughout the plant and animal kingdom, involves a wide variety of competing metals, and manifests

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Table 10. Examples of diseases associated with metal intoxication.

Intoxicant Iron Copper Zinc Cobalt Manganese Selenium Lead Cadmium Mercury Disease Hemochromatosis Wilson's disease Metal-fume fever

Heart failure; Polycythaemia Ataxia

'Blind staggers' (cattle) Anaemia; Encephalitis; Peripheral neuritis Nephritis

Encephalitis; Peripheral neuritis

either as systemic disease or as disruption of specific organ functions (Table 11). As an example, in rodents excess cadmium produces rapid tes-ticular necrosis which is prevented completely by sufficient amounts of zinco

This is a good time to recall that initial biological interest in a metal has of ten resulted from its toxic features, while an essential biological role was identified much later. Zinc was recognized to be an industrial hazard, causing 'metal-fume fever', long before its essential biological functions we re established and these are due to much lower concentrations of zinc than the phenomena familiar to toxicologists.

Table 11. Examples of conditions due to metal ion imbalances.

Organism Functional Competing Dysfunction

metal metal

Plants Mg K Growth failure

B K Chlorosis

Bacteria Fe Mn, Co, Pb Tetrapyrhole synthesis

Rodents Cu Zn Anaemia

Zn Cd Gonadal necrosis

Cattle Cu Mo Diarrhoea, Wasting

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~

- - -

It seems difficult to ever be certain th at a metal does not have an essential biological function, while it can be claimed with certainty only that -at a given level of deprivation achieved thus far - no requirement for the metal has been shown in a given species. Yet, by employing very strict metal-free conditions and controlling dietary intake over long periods before demonstrating a deficiency state, the list of 'essential' metals has grown remarkably at the expense of those thought only to be toxic. The existence of an essential biological role, of course, in no way obviates the elimination of the hazards which metals create.

Environmental redistribution of metals is a potential ecological threat, but in most instances, presently available data do not permit a credible as-sessment either of the nature of ultimately ensuing pathological changes or the time scale of their appearance. SCience, industry and government must collaborate to acquire such data. Long-term trends in environment al metal distribution must be examined and their rate of build-up must be anticipated. The effects of metals on biological systems must be studied and sensitive signals of toxicity must be found, as must be the biological consequences of long-term, low-dose metal exposure. Finally, still better, more sensitive, rapid and accurate analytical approaches are needed.

What is required most urgently is the generation of an adequate scientific base for reasoned judgments. One may hope that intense scientific efforts directed toward these problems might result in new information that could illuminate the entire field of metallobiochemistry and biology and pari passu that of environmental metal toxicity.

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148

Trace Elements and Hurnan Disease

John Lenihan

Professor of Clinical Physics University of Glasgow, U.K.

12 (1988) DELFT PROGRESS REPORT

Our meeting to-day is weIl timed. The great advances in knowledge and under-standing of the role of trace elements in science, industry, medicine and poli tics , which provide the topics of our discussions, have been achieved almost entirely during the past 40 years - the period of Professor Houtman's distinguished career. I am grateful to have the opportunity of adding my voice to the chorus of respect and admiration by which his great contribu-tions to the work of this Institute and to the wider world of science are being acknowledged.

In this talk I should like to discuss some current ideas on the role of trace elements in human disease and to mention some problems awaiting further study. These topics are not new, for speculation as to the causes of disease has always been an important component in the practice of medicine. In an-cient times as in some primitive communities to-day - diseases were attributed to supernatural causes. The Greek physicians gave attention to weather, diet, exercise and to the disturbance of equilibrium among various

internal influences or humours. In the 19th century many diseases we re shown to be caused by biological agencies, including micro-organisms and internal parasites.

Chemistry and biochemistry have become important only during the past 100 years, though there were speculations on chemical causes of diseases as early as the 16th century. Paracelsus asserted that pathology must be based on anatomia elemerita and on anatomia esserta - that is the distribution of

es-sential constituence in the body. He also taught that diseases are caused by excesses of salts and referred to cancer as morbus arsenicalis. But in the

16th century there were no scientific or technical resources to exploit these ideas in a rational way. There were empirical treatments, based on

observation; Hippocrates observed that the drinking of rusty water was

beneficial to people of excessively pale appearance. Other chemical treat-ments, based on deficient knowledge and understanding, were not so

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~ --- - - - ---

and antimony, of ten with unfortunate consequences - though some of their

ideas have persisted even into our own time.

From the 17th century onwards, the chemical composition of blood and urine were recognised as aids to the diagnosis and classification of disease. The significance of chemical substances as causes of disease was, however, ap-preciated only to a limited extent. In the absence of sensitive analytical

techniques, only diseases caused by heavy exposure to toxic substances could

be studied. In recent years a succes sion of spectacular improvements in the

techniques of analytical chemistry has made possible great achievements in our understanding of the behaviour of trace elements in health and disease.

There are two main ways in which the new knowledge has been developed and applied. The first is the molecular approach, largely developed by Dr Val lee , whose masterly review we have just enjoyed. A complementary approach us es the ideas and methods of epidemiology. This approach has been fruitful in rela-tion to a number of occuparela-tional hazards and has had a few successes in a wider context.

An important branch of trace element studies is related to geographical

variations in the prevalence of cancer. At many places in the world we find

two neighbouring communities, apparently very similar in their genetic and

demographic make-up as well as in their industrial and cultural background, but with widely differing mortality and morbidity for particular cancers. A good example is provided by some recent work of Dr Guo-Gang Hu of the Chinese Academy of Medical Sciences, who spent some time working with us in Glasgow

(Table 1).

In different parts of the Taihang Mountain area in North China the prevalence

Table 1. Molybdenum and oesophageal cancer.

Dietary intake of Mo Mortality, 1969/71 per 100,000 population Serum Mo, ng/100 ml Hair Mo, ng/g Linhsein lower 199.7

Cancer patients Controls

210 ± 30

46.4

±

4.8

220 ± 26

44.6

±

3.5

Yuhsein higher

8.56

Cancer patients Controls

290 ±

48

46.6 ±

3.8

480

± 29

(25)

of oesophageal cancer varies by as much as 100 to 1. When differences of this magnitude occur it is reasonable to look for some environmental factor which may be responsible. Trace element deficiency is one obvious possibility. In the Chinese region that I have just mentioned molybdenum has been implicated and the evidence seems quite convincing.

Analysis of the grain which forms a major part of the diet in several countries, with widely differing prevalence of oesophageal cancer, shows that the highest prevalence occurs in are as where the molybdenum content of the grain is low; the molybdenum content of drinking water in these areas is also relatively low. A similar finding has been reported from the Transkei region in Sou th Africa where oesophageal cancer is also prevalent. Dr Hu and his colleagues found that, as might be expected, the reduced dietary intake resulting fr om low levels of molybdenum in grain is reflected in urine, hair and serum. The differences in molybdenum levels in these three materials be-tween the high preval~ce and low prevalence areas are highly significant. Further experiments suggested th at plants grown on molybdenum-deficient soil have an abnormally high concentration of nitrates, providing favourable con-ditions for the synthesis of nitrosamines - which are potent carcinogens. In another experiment, the application of ammonium molybdate to molybdenum-deficient soil resulted in an increased grain output, an increase in the concentration of molybdenum in the mature grain and a decrease in the con-centration of nitrates. It was also found that the ni tros amine content of grain was significantly higher in areas with a high prevalence of oesophageal cancer.

It has been suggested that more than 80% of all human cancers have their origin in the environment. This assertion is not so dramatic as it seems, since, if we leave out infection and trauma, we might say th at all diseases have their origin in the environment. However, it does seem that a study of geographical anomalies in the prevalence of particular cancers is an impor-tant line of research and that the measurement of trace element concentrations in food and tissue may sometimes provide valuable clues. The successful development of the epidemiological approach to the role of trace elements in human disease is complicated by a number of factors:

1. The biological activities of trace elements, whether beneficialor damaging, can seldom be studied in isolation. As we have already heard, the behaviour of one trace element is of ten influenced in a decisive way by the presence or absence of other trace elements. As knowledge of these processes accumulate, it is tempting to suppose that every element interacts with every other one and that the task of sorting out such a complex pattern is beyond

(26)

~ ~ - - -

our ability. On the other hand it may be that the apparent confusion

repre-sents different aspects of some fundamental processes of interaction which has not yet been identified.

---:::;Pb

F

----Mo

- - - - W

I~I

\

As

Si

Ca

Cd

S

Hg

~J/Jg~r"~l/

J/J

I

Rb

Fig. 1. Interactions among essential and toxic elements.

Most of our knowledge of interactions among trace elements comes from animal

experiments which are of course easier to conduct and usually of greater

economic significance but there are a few observations relevant to human

disease. for instance as for copper (Tabie 2).

There is evidence that intestinal absorption of both iron and lead is abnor-mally high in subjects with low iron stores, indicated by low serum ferritin.

In Britain. some

30%

of children and menstruating women have latent iron

deficiency. shown by increased iron absorption without significant anaemia.

Table 2. Characteristics common to people with ischemic heart disease and to animals deficient in copper.

- Arterial damage - Myocardial damage - Decreased myocardial Cu

(27)

152 12 (1988) DELFLT PROGRESS REPORT

In this sizeable fraction of the population the uptake of lead from the gut may be twice the norm al level of 10%. These influences exacerbate the problem of lead poisoning in decayed urban localities, where people are vulnerable to iron deficiency, from inadequate nutrition, and to increased intake of lead, from environmental pollution.

2. The definition of an essential trace element may not be as simple as it appears. Of the 90 chemical elements which occur in nature, 14 are unlikely to be of biological significance; these are the naturally radioactive ele-ments with no stabIe isotopes and the noble gases. A further 11 eleele-ments are present as structural components in almost every tissue and occur at rela-tively high concentrations in the body. Of the remaining 65 elements, 15 are believed to be essential for animal life and therefore presumably for human life also.

But how do we know that they are essential? An element is believed to be essential if it conforms to certain conditions. An excess or deficiency should produce harmful effects which can be associated with particular biochemical changes. To this rather negative approach we can add the positive condition th at the element should take part in some identifiable biological processes necessary for the life of the animal. Since it is clearly important to know which elements are essential for the life of man, how can we find out? The experiment al approach which is quite permissible and convenient in animals is not appropriate, on ethical grounds, for the human subject. Even if this approach could be tolerated, it would not always be feasible. Many elements occur in the body and in the diet at such low levels that it would be extremely difficult to produce a dietary deficiency. This difficulty is reinforced by the fact that many elements are present in foodstuffs and in the tissues of the body at levels which are ne ar or beyond the present limits of analytical chemistry. Of course most of the elements now known to be es-sential for the life of man have achieved that status quite recently, and through the availability of highly sensitive analytical techniques.

In view of the difficulty in studying the effects of deficiency or excess in the human subject, it is worthwhile to think about indirect methods of judging whether a particular element is essential. We have been looking at this problem for a good many years in Glasgow. Our first approach - not par-ticularly clever was based on the hypo thesis that if an element is essential, it is likely to be subject to some physiological mechanism regulating its concentration in the appropriate part of the body within cer-tain limits. This process is seen clearly in relation to manganese; tissue levels remain within relatively narrow limits even though dietary intake

(28)

~ - - ~ - - - -- - -

changes by hundredfold

a large factor. In animal experiments, an increase of more than in dietary intake changed tissue levels by a factor of only two. A similar stabilising mechanism, though not so spectacular, can be identified for many other essential elements.

As the design of the body is generally economical it is reasonable to ex-peet that an element which has no biological function will not be subject to any stabilizing mechanism. It would obviously be useful to find a numeri cal index corresponding to the strictness of the regulating mechanism and there-fore presumably to the essentiality of the element concerned. One obvious way of obtaining such an index is to measure the concentration of an element in a large number of samples of a particular tissue taken from normal healthy subjects. The dispersion of the results about the mean value will be smal 1 for elements of which the concentration is closely regulated and larger for elements where no such mechanism exists. The customary measure of dispersion is the standard deviation - but it must be remembered that this number is a property of the normal distribution. Some measurements, such as serum sodium, do conform to the norm al distribution but for most of the trace elements a skew distribution is found and the standard deviation of the arithmetic mean is not a valid measure of dispersion or of anything else.

The difficulty can be overcome if, as is nearly always the case, the measured concentrations of the element concerned in a particular tissue fol-low in a log normal distribution. In this case it is appropriate to use as a measure of dispersion the geometrie standard deviation, which is the antilog of the standard deviation of log (concentration). The geometrie standard deviation can be interpreted in a rather similar way to the standard devia-tion derived from a norm al distribudevia-tion. For example, about

68%

of the observations can be expected to lie between geometrie mean multiplied by geometrie standard deviation and geometrie mean divided by geometrie standard deviation. When the test that I have just been describing is applied to con-centrations of various elements in different tissues of the body it is found th at for elements such as zinc and copper, known to be essential, the geometrie standard deviation is always less than two. For elements suspected to be non-essential, the geometrie standard deviation is usually weIl over two (TabIe

3).

In looking at these observations, my own view is that there is no sharp dividing line between essential and non-essential elements. The degree to which an element is essential depends on a number of factors, including, for example, the concentrations in the tissue concerned of other elements which

(29)

Table

3.

Geometrie standard deviation of the eoncentration of some elements in various human tissues.

Tissue Al As Hg Cu Zn Sb Aorta

2.69

2.82

3.14

1.54

1.64

Brain

2.21

2.30

3.51

1.45

1.36

Heart

2.31

2.69

3.04

1.29

1.32

2.75

Kidney

2.20

3.45

5.05

1.34

1.42

Liver

2.21

2.84

4.26

1.80

1.48

2.69

Lung

2.25

2.50

2.76

1.43

1.36

2.74

Musele

2.62

2 .

38

3.56

1.49

1.43

may be eo-operative or antagonistic. It seems also that a partieular element may be more essential in some tissues than in others. This situation ean be observed by looking at the findings for strontium which, judged by geometrie standard deviation, behaves as a non-essential element in many tissues but

behaves more like an essential element in bone; the distinetion between the geometrie standard deviation of concentration for strontium in bone and in eartilage is particularly striking (Tabie

4).

This aspect of the behaviour of strontium is presumably related to the fact that it can be fitted into the apatite structure whieh forms the hard com-ponent of bone and will indeed be taken up to a greater extent if the diet is seriously deficient in calcium. So, perhaps the concept of essential and non-essential traee elements is too simpie; we should be thinking instead

Table

4.

Geometrie standard deviation of the concentration for strontium in

various human tissues.

Tissue Sr Aorta

2.14

Brain

2.35

Heart

2.15

Kidney

1. 79

Liver

2.18

Lung

1.90

Muscle

2.77

Cartilage

3.37

Rib

1.67

Vertebra

1.45

(30)

-- ~ - - -

out a continuous spectrum of essentiality, influenced by the factors that I have just mentioned.

3.

Another source of confusion is the concept of optimal dietary intake of an essential element. In this question it is obviously reasonable to seek some guidance by estimating the intake of the element concerned in the diet of a group of apparently healthly people. Such measurements are a major source of inspiration for the committees which try to prescribe appropriate levels of dietary intake. They are, however, subject to many errors or pit-falls, as can be seen by a study of the literature.

Since estimates of daily dietary intake are so uncertain it is perhaps not surprising that recommendations of optimal intake are sometimes difficult to achieve (Appendix I). The National Academy of Science recommends a daily in-take of zinc of 15 milligrams for adults, 20 milligrams for pregnant women and 25 milligrams for lactating women. Some studies we have made in Glasgow suggest that the ave rage daily intake of zinc is only about 10 milligrams. It is, however, not easy to see how the typical Scottish diet could be modified to increase the intake of zinco An adult male, according to the National Academy of Science recommendation, needs 15 milligrams of zinc per day. The recommended total energy intake in the diet is about 11 MJ per day. The diet should therefore be made up of food which provides, on average, about 1.4 milligrams of zinc per MJ in order to obtain 15 milligrams of zinc at the same time to avoid exceeding the recommended energy intake. But the zincjenergy ratio for a typical West of Scotland diet is only 0.80 milligram of zinc per MJ (Tabie

5).

In Scotland, as in most of the world's developed countries, the white flour which is widely used looses about 80% of its zinc during the refining process. If unrefined flour was used exclusively in the diet, the necessary ratio of zinc to energy could be achieved for male adults and for children, though not for the adult women in the West of Scotland who at present receive only

7.6

milligrams of zinc per day in a typical family diet. The replacement of white flour by wholemeal flour is not arealistic possibility; nor is the only other possibility, which is the consumption of about

3

kilograms of cab-bage per day, to reach the recommended zinc-energy balance.

It seems therefore that the diet in the West of Scotland - and presumably in other parts of industrial Britain - should be supplemented by inorganic zinc or that the recommended daily allowance should be reconsidered. It must of course always be remembered that measurement of the total amount of a par-ticular element in food or tissue is not the whole story. In many Middle

(31)

156 12 (19BB) DELFT PROGRESS REPORT Table

5.

Zinc, ave rage daily intake in Glasgow Area.

mg Zn MJ mg Zn/MJ Meat 2.97 1.66 1.79 Cereal products 2.08 3.56 0.58 Dairy products 1.68 2.41 0.70 Vegetables 0.68 0.97 0.70 Beverages 0.26 0.12 2.17 Other items 0.93 2.05 0.45 Daily intake 8.6 10.76 0.80 Recommended intake 15 11 1.4

Eastern countries the daily intake of zinc reaches the recommended level of 15 milligrams, yet effects of zinc deficiency are quite common. The problem here is that diets consisting mainly of unleavened bread and beans are rich in phytates which bind the zinc and greatly reduce its absorption.

These difficulties are of course related to the distinction. not always fully appreciated, between intake and uptake. It would clearly be much more useful to measure the uptake of an element - that is the total amount absorb-ed from the gut and the lungs - rather than the intake which is the total amount ingested or inhaled. This problem is complicated by the fact that up-take is seldom proportional to inup-take. For lead, an element of great concern at the present time, the uptake appears to be roughly proportional to the cube root of the intake. Consequently the elimination of various sources of lead in the environment will not always make a very significant difference in the concentration of lead in the blood, which is the significant parameter in judging the biological effects. The control of lead and other environmental hazards is a ·topic to which we shall probably give more attention later in

this meeting.

4.

The great advances in analytical chemistry which have occurred in recent years have led to a situation in which it was much easier to produce answers than to formulate meaningful questions. A great deal of effort has been wasted by asking the wrong questions. For many years, experimenters have been trying to find differences in the concentration of various trace ele-ments between tumour tissue and normal tissue. These investigations are of course made in the hope of finding a clue to the cause of cancer. It is cer-tainly true that some uncommon occupational cancers are associated with

(32)

~ - - -- - -

excessive exposure to particular elements or compounds. In the study of the more common cancers, it is not difficult to demonstrate differences in chemi-cal composition between tumour tissue and normal tissue. It now seems, however, that there is a futile quest. Recent work has shown that the dif-ferences in chemical composition between tumour cells and normal cells are quantitative rather than qualitative. The transformation of a normal cell into a malignant cel I seems to be accompanied by the overproduction of proteins and other substances which are normal constituents of the cello It seems therefore that differences in trace element concentrations are for-tuitous and do not in fact convey any useful information.

In looking over the contemporary scene, Professor Houtman may fee I that the problems to be attacked in the future are more numerous and more difficult than those which attracted him to the study of trace elements at the start of his career. He may feel that his own work has created more problems than it has solved. But that is a welcome state of affairs, for a science that solves all its problems is of no further interest or use. Professor Houtman's con-tribution to science resides not only in what he has done but in the challenging problems that he has uncovered for those who will follow in his path, here and in all the other laboratories which have been touched by his inspiration.

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158 12 (1988) DELFT PROGRESS REPORT APPENDIX I

Daily dietary intake. pg

1. SELENIUM 62 18-26 60-150 113-220 137 2. MERCURY 20 4-20 1-31 10 3.9 58 3. ARSENIC 70-170 400 900 140 55 4. MANGANESE 7 3.7 2.8 2.5-3.2 2.7 Schroeder et al (1970) Robinson et al (1973) Thompson et al (1975) Cross et al (1979. unpublished) Gibbs et al (1941) Westlöö (1965) Norden et al (1970) (u.S. hospital) (UK) (North-Eastern U.S.) (Canada) (Scotland) (u.S. ) (Sweden) (Sweden) U.K. Ministery of Agricult.(1971) (UK) Mahaffey et al (1975)

Cross et al (1979)

Nakao (1960)

Schroeder and Balassa (1966)

Duggan and Lepscomb (1969) Cross et al (1978)

Monier-Williams (1949) North et al (1960) Murakami et al (1965) McLeod and Robinson (1972) Hamilton and Minski (1973)

(U.S.) (Scotland) (Japan) (US) (US) (U.K. ) (Scotland) (U.K. ) (U.S.A. ) (Japan) (New Zealand) (U.K. )

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- ~ - - -

-DELFT PROGRESS REPORT (1988) 12

Sou rees

and Pathways of Traee Elements in the Environment

J.P.W. Houtman

Professor of Radiochemistry, Interuniversity Reactor Institute and The University of Technology, Delft, The Netherlands

159

My task for today - to provide some relevant information about sources and pathways of heavyelements in our environment - is not an easy one, though I admit that somebody who has access to the facilities of a nuclear reactor institute has ample possibilities to grow into that subject. For the outsider it is useful to explain why this is so. The presence of a nuclear reactor of 2 MW power - as we have here in Delft - enables the determination of metals at the very low concentrations in which they are present in the environment. Moreover, experience in handling and measuring radioisotopes stimulates tracer experiments which give insight in physiological pathways of the metals in plants,

basis of bet ter to

animals and man. Thus, it was natural th at our institute on the interuniversity cooperation became involved in this type of work, say, the staff engaged in various aspects which do not of itself form a complete pattern of environmental science. Therefore, you will now understand that I have some difficulties in composing a reasonable complete and coherent picture, also because it is a vast extending field with many aspects, but at the same time with an enormous amount of vacancies in our knowIedge. As part of a general survey of the field I will provide some details selected from literature and from IRI work.

Trace elements are important for our health and thus also for society. Why this is so and on what fundamental basis it is stooled, has been explained by prof. Val lee and prof. Lenihan. One distinguishes between essential and toxic elements, though some essential elements could under certain conditions also be toxic. This depends on chemical form and concentration. Moreover, there are not only dangers of surfeit but also of deficiency. Because of that, it seems bet ter for today not to treat the subject in general terms but to con-centrate on certain problem elements. At the same time I will formulate our problems as the long term ones so that I will not speak about acute cases of poisoning of shortages.

On the basis of present day information it seems reasonable to de fine as problem elements cadmium, lead and mercury as far as we are concerned with overdose, and iodine and fluorine if also deficiencies are to be treated. In both categories there are some elements of secondary concern. Overdoses have

(35)

been reported for arsenic, cobalt, chromium, manganese, molybdenum, nickel,

thallium and tin though some of them are also essential. Problems with them

can appear also in larger population groups, but as far as we know now only

under special conditions, mostly as a result of uncontrolled industrial activities. In the second category there seem to be some elements of which we

are becoming aware of long run detrimental influences of deficiency. These are copper, magnesium, selenium and zinco The information is still rather

vague but becomes more and more indicative, for instance in relation with

cardiovascular diseases. It may weIl be so that in a few years a conference

like this would be justified with respect to these problems. Effects of

un-derdosage of iodine and fluorine are already well-known. More than one

counter-measure is available which for our country has left some space for

political maneuvering. For this symposium, our primary interest goes out to

the elements cadmium, lead and mercury. The reason why I have selected these

elements becomes clear from Table 1, in which estimated real intakes in the

Netherlands are compared with the limits set by FAO/WHO.

Table 1. Intake per head of population (in pg/day).

Element

Cd

Pb Hg As

Max. Permiss. Intake FAO/WHO

60-70 460

46 3250

Estimated Real Intake The Netherlands Range Medium <15-80 20 10-1460 80 5-92 5 <11-1000 22

Though median intakes are still far from the maximum permissible intake (MPI) values, the ranges observed indicate that these MPI values are probably

passeq by a percentage of the population which cannot be neglected. Moreover,

MPI values are constantly under criticism and have the tendency to drop.

In Table 1 is also introduced arsenic, because some time ago this element

was also looked upon as critical and thus was monitored in foods and

Heavy Metals in the Environment: A threat to Public Health?

Heavey Metals in the Environment. A threat to Public Health?

Contents

The Metallobiochemistry of Zinc and Cadmium

c

N

0

10

2

r

w

>

~

W

w

IÖ

2

U

z

0

Z

~

m

(3-4

T

~

0

a..

0

0

METAL CONCENTRATION

)

3).

3.

4).

4).

4.

3).

His 63

His 93

i f

3.

4).

CYS97

4.

6.

6.

7).

7.

8)

8.

6,500

7,000

,

30-34

%

60

61

5),

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s

s

s" /

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Cd_ S

I /

I /

---r-Cd -

S

s,

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S

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Cd

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₁₀

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