Agents and Actions vol. 6- 1/2/3 (1976) Birkh~iuserVeflag, Basel

Ambivalent Role of Copper in Inflammatory Disorders M.W. Whitehouse Department of Experimental Pathology, John Curtin School of Medical Research, The Australian National University, Canberra, ACT, 2600, Australia

Abstract Soluble acute/chronic agents in rats.

copper irritants

(Cu) and

preparations are both effective anti-inflammatory

Copper is a prevalent component in several folk remedies for arthritis. Patients with rheumatoid arthritis and ankylosing spondylitis are reported to have higher-thannormal levels of serum copper, mainly associated with albumin. The anti-arthritic drug, D-penicillamine (Pn), efficiently strips Cu from some of its (pharmacologically inert) storage forms, e.g. Cu-albumin, Cu-polynucleotides yielding low M.W. Cu-Pn complexes, which show anti-intlammatory activity (ca. 5X phenylbutazone) in rats irritated with carrageenan, oleyl alcohol, sodium urate and adjuvants. Under certain conditions Pn also blocks the amine-oxidase activity of caeruloplasmin, a circulating copper protein which is elevated in inflamed animals (an 'acute phase reactant'). Drugs, nutritional factors and the disease process may all possibly affect the movement of copper in vivo between inert ~ pharmacoactive e-~ toxic forms. Results and discussion This p r e s e n t a t i o n is p r i m a r i l y a review o f some o b s e r v a t i o n s a n d practises which on the one h a n d relate to i n f l a m m a t o r y disorders a n d on the o t h e r h a n d i m p l i c a t e an i n v o l v e m e n t o f copper. T h e references given here will allow

i n t e r e s t e d r e a d e r s to e x a m i n e the o r i g i n a l d a t a m o r e critically. T h e T a b l e s u m m a r i z e s s o m e o f this d a t a or the conclusions that m i g h t be d r a w n therefrom. A s s o c i a t i o n of c o p p e r with i n f l a m m a t o r y d i s e a s e T w o h u m a n diseases i n v o l v i n g j o i n t destruction are certainly c h a r a c t e r i z e d b y h i g h levels o f s e r u m copper. T h e y are r h e u m a t o i d arthritis [1] a n d a n k y l o s i n g s p o n d y l i t i s = Bechterew's disease [2]. Both these diseases r e s p o n d to D-penicillamine, a c o p p e r - m o b i l i s i n g d r u g [3, 4]. H y p e r c u p r e m i a does not seem to b e n o t a b l y associated with o t h e r forms o f arthritis. Also, p e n i c i l l a m i n e does not s e e m to confer a n y b e n e fit in o t h e r i n f l a m m a t o r y disorders, especially o t h e r types o f arthritis. O n e characteristic o f severe i n f l a m m a t i o n is the presence o f the so-called 'acute p h a s e r e a c t a n t s ' in the serum. These i n c l u d e e l e v a t e d levels o f fibrinogen, several g l y c o p r o t e i n s a n d the c o p p e r - c o n t a i n i n g p r o t e i n c a e r u l o p l a s m i n . M e a s u r e m e n t s o f these proteins h a v e a f f o r d e d some correlations with the progress or r e m i s s i o n o f the u n d e r l y i n g disease p r o v o k i n g an i n f l a m m a t o r y response, b o t h in m a n a n d a n i m a l s .

Some relationships between copper (Cu) and inflammation/arthritis. Disease





Elevated serum Cu: (?) Normal caeruloplasmin (?) 'Extra' Cu on Albumin

Treat disease with: Cu bangles Cider vinegar Shellfish Mushrooms Nuts

Cu(II) is anti-intl.

I)-Penicillamine = antirheumatic (mobilizes Cu?) Hepatocuprein = Orgotein is 'anti-intl.'

Cu(II) = irritant

Cu complexes of anti-intl, drugs are (?) more potent than A.I. drugs alone

Oedema Counter-irritants? Granuloma Counter-irritants? (natural anti-intl.)

202 One report [5] has clearly 'identified the caeruloplasmin level as being not much greater than normal in patients with rheumatoid arthritis. The excess copper in the serum of these patients is therefore associated with another serum component, believed to be primarily albumin. Albumin-bound copper seems to be a ciruculating inert store of copper that is certainly devoid of pharmacological activity. What function this extra copper may have in promoting inflammatory disease is at present unknown. Where excess albumin catabolism occurs, as for example in an inflamed tissue where both tissue destruction and tissue repair may be considerably in excess of normal, some of this extra copper could be 'off-loaded' causing either copper-mediated tissue suppression or copper-incited tissue irritation or even both simultaneously. Caeruloplasmin may have a protective role by virtue of its ability to oxidise certain amines [6].

Anti-inflammatory activity of copper salts In conventional assays employing small animals, cupric salts show anti-oedemic and anti-erythemic activity and may inhibit the granulomatous response to implanted foreign materials [7-10]. Sorenson [8, 9] has claimed that copper salts of many acidic anti-inflammatory drugs are more potent than the parent acids in some anti-inflammatory models. Others have disputed this claim. There is a technical problem that many of these copper salts or chelates of copper with other organic compounds (including non-acidic anti-inflammatory agents, e.g. steroids) are very insoluble. Care must be taken that they are not administered in vehicles, such as carboxymethylcellulose, which might in turn render their copper content further unavailable. Even though preformed exogenous copper salts may show only modest drug activity in some assays, we must still carefully consider Sorenson's hypothesis that anti-inflammatory drugs could react with available copper in vivo to generate more effective inflammation-regulating copper complexes. Stated in another way, the hypothesis suggests that the conventional anti-inflammatory drugs are perhaps only vehicles for mobilising or translocating pharmacologically-inert copper to engender pharmacoactive forms of copper. This may perhaps be an extreme exposition of Sorenson's hypothesis but it certainly seems to describe how penicillamine

M.W. Whitehouse might for example confer benefit in rheumatoid disease (see below).

Association of copper with anti-rheumatic therapies A number of folk remedies for arthritis happen to involve sources of copper. Some of these are foodstuffs, such as shellfish or nuts, known to contain rather more copper than many other items in the average Occidental diet. Another 'remedy' recently popularized in the paperback books of a Vermont physician, D.C. Jarvis, is cider vinegar - a product that, if prepared in the traditional mode of aging apple juice in copper-lined vessels, would almost certainly contain copper ions as a result of the effects of the acids in the juice (e.g. malic) leaching minute amounts of the copper vessel. Copper jewellery, especially bangles, has enjoyed a considerable vogue in do-it-yourself 'treatments' of arthritis. This efficacy of the bangle has been linked with the degree of green stain formed where the bangle is in contact with the skin. It is certainly difficult to comprehend how the topical application of copper salts of sebaceous acid components could be so effective. Nevertheless we cannot simply dismiss the bangle as so much 'ballyhoo' (especially in view of the improbability that very much copper could be absorbed through normal dermal tissue) while there is definitive evidence that parenterally administered copper preparations can inhibit many inflammatory responses in animals. At this stage we must ask the blunt question 'Are the higher than normal levels of copper in the serum of patients with rheumatoid arthritis due to the disease per se, or to selfprescribed copper-rich remedies?' As noted above, D-penicillamine (fl-mercaptovaline) is an effective anti-rheumatic drug. It is able to 'strip' albumin-bound cupric (CuII) ions from the natural copper-albumin complexes [10, 11] by (i) substituting for a second albumin molecule or any one of the natural amino acids which form the natural ternary complexes, and (ii) reducing the Cu(II) to Cu(I). The Cu(I) so formed then dissociates from the prime Cu(II)-binding site on the albumin molecule since the groups which bind Cu(II) strongly (e.g. amino, imidazoyl, peptide bond) all have low affinity for cuprous ions.

Ambivalent Role of Copper in Inflammatory Disorders

Alb-Cu(II) - X + Pn ~ Alb-Cu(II) - - Pn+X high M.W. Cu Alb + Cu(I) + oxidized Pn. low M.W. Cu (X = L-aminoacids or albumin) Elsewhere we have presented data indicating that whilst the copper-albumin complexes have no anti-inflammatory activity in acute assays, the various low M.W. form of Cu(I) and Cu(II) liberated from the complexes by penicillamine are indeed pharmacoactive [10]. It must be emphasized that penicillamine can only generate such activity if there is an endogenous pool of latent (pharmaco-inert) copper in vivo. This may not occur significantly in normal individuals but is very likely in patients with hypercupremia and minimally elevated caeruloplasmin levels. These conditions seem to be met in patients with rheumatoid arthritis or ankylosing spondylitis. Preparations of superoxide dismutase are currently undergoing trials for the treatment of arthritic disorders. One such preparation (Orgotein) is obtained from beef liver and is a protein containing both copper and zinc [121. The copper content is so characteristic of this enzyme that it was known as hepatocuprein, or erythrocuprein, or cerebrocuprein (depending on tissue source) and believed to be merely a tissue storage form of copper, until its enzyme function was discovered only very recently [13].

Irritancy of copper preparations Simple cupric salts and copper complexes (e.g. with glycine, citrate) are highly irritant and elicit considerable oedema and subsequent granuloma development, or ulceration, at their site of injection into rats. By contrast copper-albumin complexes and the mixed valency complexes formed when penicillamine strips copper from albumin, are almost devoid ofirritancy. The question arises whether the anti-inflammatory effect of many copper salts is not due to a counter-irritant phenomenon [7]. We have certainly noted that the injection of copper salts into a rat paw causes the fairly rapid appearance of 'acute phase reactants' in the serum (within 24-28 hours), which almost certainly include anti-inflammatory proteins [ 14].


Some conjectures (1) In addition to these anti-inflammatory proteins, low M.W. endogenous anti-inflammatory substances have been described which have a high affinity for albumin or other plasma prteins [15, 16]. It is noted in some reports describing these substances that they are 'less available in sera from rheumatoid patients, even though they are still present in adequate quantity. The inference is that they are bound to a higher degree in rheumatoid sera, particularly to albumin, than in the sera of normal individuals. Perhaps the extra copper associated with albumin of rheumatoid subjects causes excessive binding of these agents, compared with their binding to the copper-poor albumin of normal individuals. If this is true, then an appreciable amount of albumin-bound copper might be truly pathognomonic for inflammatory disease (as well as for copper intoxication) because it effectively withholds natural homeostatic anti-inflammatory principles from being released from a circulating storage form. Intervention with penicillamine could then release not only pharmaco-active copper but perhaps also help to unlock other hitherto unavailable factors, associated with albumin, which can modulate inflammation. (2) The poor response of some patients with rheumatoid disease to penicillamine therapy might be due to lack of 'extra' copper, i.e. not associated with caeruloplasmin. (It should be noted that copper contained in caeruloplasmin is not normally available for exchange with extrahepatic copper or accessible to simple copper-binding agents, in contradistinction to albumin-bound copper which is accessible.) This might particularly be so in those patients who, rather surprisingly, after successfully responding to this drug for 2 years or more, no longer seem to benefit from further treatment with penicillamine and actually suffer recurrence of their disease. One might anticipate that eventually all the 'surplus' copper might be 'washedout' of these patients by the drug. So if the drug is not continually active per se but is only effective in combination with endogenous (latent) copper, then a rebound must be expected if no steps have been taken to keep the patient loaded with copper, e.g. denying the use of a copper bracelet. This speculation can at least be scrutinized fairly rigorously by determining the non-caeruloplasmin copper levels in


patients throughout a course of penicillamine treatment and noting what association there is, if any, between the available copper in serum and the patient's responsiveness to the penicillamine. (3) Does the high female-male ratio seen in rheumatoid arthritis have anything to do with the fact that normal females have higher serum copper levels than normal males [1]? (4) Spontaneous remissions of adult/juvenile rheumatoid arthritis occur with obstructive jaundice/liver disease or pregnancy; i.e. conditions in which the serum copper levels are frequently elevated above the normal range [ 17, 18]. A considerable proportion of the body's copper burden is normally excreted via the bile [19, 20] so that in obstructive jaundice the biliary copper must be redistributed elsewhere within the body. Are these natural remissions of arthritis (with spontaneous hypercupremia?) further pointers to the efficacy of copper as an inflammalytic regulator?

Reconciling the irreconcilable We are often prone to believe that a single agent has a single action, be it black or white. The title of this communication emphasizes that this may not be so and that we should be prepared to accept that copper, at least, may be both injurious and beneficial, even at one and the same time. Many drugs actually injure the gastric or intestinal mucosa but still confer systemic anti-inflammatory activity. For certain drugs for which this is true, we can now understand this particular paradox in terms of a single mechanism of action at the molecular level, namely inhibition of prostaglandin synthesis. For other drugs, e.g. steroids, we still lack such a unifying hypothesis to reconcile the toxic/beneficial effect in different compartments. This has not prevented, however, widespread acceptance of the steroids as very useful anti-inflammatory agents. As yet there seems also to be no acceptable unifying hypothesis to explain why copper should be both injurious, perhaps pathognomonic for inflammatory disease and also prove beneficial in regulating an inflammatory response. Why this should be a property of copper, rather than other metals, is also a mystery at the present time. In place of further speculations, the attention of both pharmacologists and pathologists and pathologists alike might be more usefully engaged in studying factors which determine

M.W. Whitehouse

the distribution and redistribution of copper between the following 3 compartments: Inert

Ambivalent role of copper in inflammatory disorders.

201 Agents and Actions vol. 6- 1/2/3 (1976) Birkh~iuserVeflag, Basel Ambivalent Role of Copper in Inflammatory Disorders M.W. Whitehouse Department...
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