Age pigments and free radicals: fluorescent lipid complexes formed by iron- and copper-containing proteins

144

Biochimica et BropFysica Acta 834 (1985) 144-148

Elsevier BBA 51877

Age pigments and free radicals: fluorescent lipid complexes formed by iron- and cop~r-containing proteins John M.C. Gutteridge Division of Antibiotics and Chemistry National Institute for Biological Standards and Control, Holly Hill, Hampstead, London NW3 6RB IU. K.1

(Received November 5th, 1984)

Key w3rds: Lipid peroxidation; Free radical; Desferrioxamine; Transition metal; Hydrox~ero~de; Fluorescent lipid complex

Aging;

Haem and non-haem iron-containing proteins stimulate lipid peroxidation with the fo~ation of fluorescent lipid compiexes. This process requires the presence of Iipid hydroperoxides which release ferrozine-reactive iron from haem-containing proteins. Stimulation of lipid peroxidation by the released iron is inhibited by the iron chelator desferrioxamine. Copper ions, although more stimulatory towards fluorescent lipid complex fo~ation than iron ions, do not stimulate lipid ~roxidation when tightly bound at the active centre of proteins, but are reactive when loosely bound to albumin and histidine.

Inaction Age pigments arise in normal and prematurely ageing tissues by the interaction of oxidized lipid and cellular proteins. These polymeric lipopigments have been classified by their colour, fluorescent properties, solvent solubulity and chemical composition, resulting in descriptive terms such as pre-ceroid, ceroid, and lipofuscin. (For a review see Elleder [l].) Experimental animal studies have shown that vitamin E-deficient or oxidatively stressed animals increase their cellular deposits of lipofuscin, suggesting a role for free radical-mediated oxidation of lipid [Z]. In human pathology, lipid oxidation (peroxidation) is frequently considered a mechanism by which free radicals damage cells and organelles. However, in many cases lipid peroxidation is no more than an inevitable consequence of cell damage [3], consistent with the known fact that damaged cells peroxidize more readily than normal cells. When damaged, cells release metalcontaining proteins from protected sites within the ~5-27~/85/$03.30

cell and these react with lipid peroxides to stimulate the process of lipid peroxidation. In this context, lipid peroxides should be considered as normal lipid metabolites and not exclusively products of non-enzymic free radical processes. Transition metal ions, particularly iron and copper, are the most likely catalysts of lipid peroxidation in vivo f41. In this present study the ability of iron- and copper-containing proteins to stimulate the formation of fluorescent lipopi~ents in phospholipid membranes has been investigated as a model for in vivo pigment accumulation. Methods and Materials Catalase (bovine liver thymol-free), xanthine oxidase grade I, ferritin (horse spleen), cytochrome c (horse heart), caeruloplasmin (human type III), dopamine-&hydroxylase (bovine adrenals), albumin (human fatty acid free), terr-butyl hydroperoxide, linolenic acid and Lubrol ‘PX’ were from Sigma Chemical Co. Methaemoglobin was

D 1985 Elsevier Science Publishers B.V. (Biomedical Division)

145

from Calbiochem. Peroxidase (horseradish) was from Hughes and Hughes Ltd. and desferrioxamine (‘Desferal’) from Ciba-Geigy. All other chemicals were of the highest purity available from BDH. Reagents were prepared in distilled water treated with Chelex resin to remove trace metals. Lipid peroxidation. Multilamellar liposomes were prepared in 0.15 M NaCl (pH 7.4) as previously described [5]. Before use they were treated with Chelex resin (0.5 g per 10 ml) to remove metal ions, and centrifuged at 3000 X g for 10 min to deposit the resin. 0.2 ml of liposomal suspension were added to 0.2 ml phosphate-saline buffer (pH 7.4) (0.1 M Na,HPO,/NaH,PO, in 0.15 M NaCl). 0.1 ml of iron salts, copper salts or proteins were added at this stage (for final reaction concentrations see relevant Tables and Figs.) and the tubes incubated at 37°C with appropriate controls and blanks for the times indicated. The iron chelator desferrioxamine was added, where indicated, before the addition of the metal catalysts. Measurement of lipid peroxidation. At the completion of incubation, 1.0 ml of Lubrol (1 SE:w/v) was added to each tube and fluorescence measurements were taken at excitation 360 nm and emission 430 nm. Results were expressed in relative fluorescence intensity units using a reference standard of tetraphenylbutadiene (lo-’ M). Release of iron from methaemoglobin by hydroperoxides. The ferrozine method of Carter [6] was

used to measure non-haem iron. Linolenic acid hydroperoxide was prepared as previously described [7]. Methaemoglobin 1 mg/ml was in-

cubated in phosphate buffer (pH 7.4) with equal volumes of linolenic hydroperoxide or t-butyl hydroperoxide for 2 h at 37°C at the final concentrations shown in Fig. 4. Non-haem iron released from methaemoglobin was determined by its reaction with ferrozine and measured at 562 nm. All the experiments shown are the mean of three separate assays which differed by less than * 6%. Results Haem-iron-containing proteins (catalase, methaemoglobin, peroxidase and cytochrome c) and non-haem-iron proteins (ferritin and xanthine oxidase) when added to phospholipid liposomes stimulate the formation of fluorescent lipid complexes (Table I). As previously observed [4,5] cupric salts were more stimulatory towards fluorescent complex formation than iron salts. This stimulatory property of copper is maintained when copper is loosely complexed with albumin or histidine but not when copper is tighly bound at the active centre of proteins like caeruloplasmin and dopamine+-hydroxylase (Fig. 1). Heat denaturation of these copper-containing proteins appeared to release some of the tightly bound copper and increased their stimulatory activity by 16% towards fluorescent lipopigment formation (data not shown). Ferric salts, although less active than ferrous salts, stimulate the formation of fluorescent lipopigments [4,5]. Similarly, ferric ion-containing

TABLE I FORMATION OF PHOSPHOLIPID FLUORESCENCE

STIMULATED BY HAEM AND NON-HAEM IRON PROTEINS

Final protein concentrations were 0.02 mg/ml. Fluorescence was measured at excitation 360 nm, emission 430 nm, and expressed as relative fluorescence intensity units. Incubations were performed at 37°C. Incubation time (mm)

FeCl, (0.6 mM)

Catalase

Methaemoglobin

Peroxidase

Fenitin

Cytochrome c

Xanthine oxidase

0 30 60 90 120 150 180

12 13 14 16 17 19 21

12 13 15 17 18 20 21

12 18 25 33 41 50 51

12 16 17 21 24 27 31

12 13 14 15 16 17 18

12 12 14 14 15 16 18

12 13 15 16 17 17 19

146

,4%

175r

,/ ,’

150

125t

501

m c : 100

2

G a

k

: 40

75

I 30;

50

25

60

90

120

.-Al

150

180 mIP

Fig. 1. The effect of copper-containing proteins and copper complexes on fluorescence formation at 430 nm, following excitation at 360 nm. Concentrations shown are final reaction concentrations. l, copper salt 0.6 mM; 0, copper salt 0.6 mM+ albumin, molar ratio 1: 300; 0, copper salt 0.6 mM+ albumin, molar ratio 1: 1; n, copper salt 0.6 mM+histidine molar ratio 1: 1.2; V, caeruloplasmin, 0.02 mg/ml; V, dopamine-fi-hydroxylase, 0.02 mg/ml. RFI, relative fluorescence intensity.

30

60

90

120

150

180

mi”

210

Fig. 3. Inhibition of haem-iron-stimulated fluorescence formation by desferrioxamine. Final reaction concentrations are shown. 0, methaemoglobin 0.02 mg/ml; 0, methaemoglobin 0.02 mg/ml+ desferrioxamine 0.7 mM; W, ferric salt 0.6 mM; 0. ferric salt 0.6 mM + desferrioxamine 0.7 mM. RFI. relative fluorescence intensity.

proteins, in the form of haem-iron, stimulate fluorescent lipopigment formation (Fig. 2). Their stimulatory activity can also be increased by heatdenaturation (Fig. 2). A substantial proportion of the lipid fluorescence formed by haem-iron-conmining proteins can be inhibited by the addition of desferrioxamine, suggesting release of complexable ferric ions from the proteins during incubation with lipid. Fig. 3 shows inhibition of methaemoglobin-stimulated formation of fluorescent lipid products by desferrioxamine. Similar results were obtained with peroxidase and cata-

Fig. 2. The effect of haem-iron proteins on fluorescence formation at 430 nm, following excitation at 360 nm. Concentrations shown are final reaction concentrations. Proteins were heat-denatured at 100°C for 10 min. 0, methaemoglobin 0.02 mg/ml; l, methaemoglobin heat-denatured; 0, peroxidase (horseradish) 0.02 mg/ml; n, peroxidase (horseradish) heat-denatured; A, catalase 0.02 mg/ml; A, catalase 0.02 mg/ml heat-denatured; x, cytochrome c 0.02 mg/ml; +, cytochrome c 0.02 mg/ml heat-denatured. RFI, relative fluorescence intensity.

147

numerous aldehydic malondialdehyde. 020

Fe and Cu I

2LOOH

-+

fragments,

including

LO’ +LO; +H,O

complexes

Maloidialdehyde

Fig. 4. Release of ferrozine-reactive iron from methaemogfobin, following incubation with hydroperoxides. Methaemoglobin concentration, 0.14 mg/ml. Incubation time, 2 h at 37°C. Final reaction concentrations are shown. l, linolenic hydroperoxide; n, tertiary butylhydroperoxide; A, linolenic hydroperoxide added to methaemoglobin at the end of the incubation time before measurement of ferrizine-reactive iron (control).

lase, resulting in inhibition by desferrioxamine of 60 and 50%, respectively (data not shown). To test the assumption that lipid peroxides were responsible for the release of iron from haem proteins, methaemoglobin was incubated with different concentrations of linolenic hydroperoxide and t-butyl hydroperoxide. Released iron was measured by its reaction with ferrozine. Fig. 4 shows that iron was released from methaemoglobin in amounts proportional to the concentration of added hydroperoxide. Discussion

Lipid peroxidation in bulk lipids is characteristically a free radical chain reaction initiated by the abstraction of a hydrogen atom from a polyunsaturated fatty acid side-chain. The resulting carbon-centred radical reacts rapidly with dioxygen to form a peroxyl radical (LO;) able to propagate the chain reaction by further abstraction of hydrogen. This results in the formation of relatively stable lipid hydroperoxides (LOOH). In the presence of suitable transition metal ion catalysts these peroxides decompose to yield alkoxyl radicals (LO’) and peroxyl radicals (able to initiate further peroxidation, Eqn. 1) as well as

To explain the occurrence of lipid peroxidation in biological systems, considerable emphasis has been placed on the formation of an initiating species such as the hydroxyl radical (OH ‘). However, since lipid peroxides are products of lipid metabolism, it is likely that transition metal complexes play a major role in stimulating peroxidative sequences in vivo. Indeed, lipid peroxidation and age-pigment formation is unlikely to occur without the presence of iron and copper complexes [4] and these metal ions have been found in high concentrations within the lipopigments [9-121. Non-protein-bound iron, protein-bound haem and non-haem iron have been shown to stimulate lipid peroxidation [13,14] by decomposition of lipid peroxides. Indeed, an early observation by Hartroft [15] noted that pigment resembling ceroid was formed in vivo anywhere at sites of intimate mixing of unsaturated fatty acids and red blood cells. Ferrous salts, however, under carefully controlled conditions can directly initiate lipid peroxidation by generating OH’ radicals [16,17]. Low-molecular-weight iron and copper complexes able to catalyse both OH’ radical formation and lipid peroxide decomposition have recently been detected in samples of human cerebrospinal fluid, synovial fluid, serum and sweat [l&19]. In the present study it has been shown that iron tightly bound to haem and non-haem iron proteins can stimulate the formation of fluorescent lipid complexes. Heat denaturation of these proteins, with release of metal complexes, further stimulates this process. An interaction between haem-iron proteins (the iron of which does not react with ferrozine) and lipid hydroperoxide appears to release ferrozine-reactive iron from the proteins. Similar stimulatory properties have previously been seen with the non-haem iron protein, ferritin [8]. Since desferrioxamine, a powerful inhibitor of iron salt-stimulated lipid peroxidation [20], substantially inhibits haem-iron-dependent formation of fluorescent lipid complexes, it suggests that the iron complexes released from haem-iron proteins

148

play an important role in stimulating lipid peroxidation. Copper salts can be considerably more stimulatory towards the formation of fluorescent lipid complexes than iron salts [4]. However, copper tightly bound to proteins like caeruloplasmin and dopamine-P-hydroxylase does not stimulate lipid peroxidation, whereas copper loosely bound to albumin and histidine can do so. Fluorescent lipopigments, whether of the Schiff-base type [21] or of polymalondialdehyde [22] require suitable metal catalysts for their formation. It can be concluded that iron and copper salts, complexes in which iron or copper ions are loosely bound and proteins in which iron ions are tightly bound can stimulate fluorescent lipopigment formation. Acknowledgements

JMCG is grateful to Al&air Beard and Gregory Quinlan for their technical assistance. References 1 Elleder, M. (1981) in Age Pigments 204-230, 2 Wohnan, 265-275,

(Sohal, R.S., ed.), pp. Elsevier/North-Holland, Amsterdam M. (1981) in Age Pigments (Sohal, RS, ed.), pp. Elsevier/North-Holland, Amsterdam

3 Halliwell, 139661397 4 5 6 7

B.

and

Gutteridge,

J.M.C.

(1984)

Lancet

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