Binding of manganese in human and rat plasma

Biochimica et Biophysica Acta 840 (1985) 163-169 Elsevier

163

BBA 22049

B i n d i n g o f m a n g a n e s e in h u m a n a n d r a t p l a s m a A.M. Scheuhammer

* a n d M . G . C h e r i a n **

Department of Patholog)', Health Sciences Centre, University of Western Ontario, London, Ontario, NrA 5C1 (Canada) (Received November 12th, 1984) (Revised manuscript received March 5th, 1985)

Key words: Manganese binding; Transferrin; Albumin; (Blood plasma)

Albumin, transferrin and 'transmanganin' have all been proposed as the major Mn-binding ligand in plasma. The present investigations were initiated in order to resolve these discrepancies. Compared to other metals tested (l°gCd2+, 65Zn2+, 59Fe3+), S4MnZ+ bound poorly to purified albumin. The addition of exogenous albumin to plasma did not result in an increased 54Mn radioactivity associated with this protein. Also, incubation of 6SZn-albumin in the presence of excess Mn 2+ (1 mM) did not result in the displacement of Zn from albumin or Mn binding. In contrast to these results, 54Mn was bound to purified transferrin, not as readily as F e 3+, but better than Z n 2+ o r C d 2+. Saturation of transferrin with F e 3+ (1.6 pg F e / m g ) prevented the binding of S4Mn indicating that Mn probably binds to Fe-binding sites on the protein. Polyacrylamide gel electrophoresis further demonstrated the association of 54Mn with transferrin r~lther than with albumin in both human and rat plasma. The amount of s4 Mn radioactivity recovered with transferrin increased as incubation time was increased, probably due to oxidation of Mn z+ to Mn 3+. Mn binding to transferrin reached a maximum within 5 and 12 h of incubation. About 50% of S4Mn migrated with transferrin, whereas only 5% was associated with albumin. A significant portion (20-55%) of the 54Mn radioactivity migrated with electrophoretically slow plasma components whose identity was not determined. Possibilities include a2-macroglobulin, heavy ,/-globulins a n d / o r heavy lipoproteins.

Introduction The fact that manganese (Mn) is both an essential trace element [1,2] and a potential neurotoxicant which selectively affects the extrapyramidal motor system in humans [3-5] has stimulated considerable scientific study aimed at clarifying the mechanisms by which Mn is absorbed, distributed,

* Present address: Wildlife Toxicology, Canadian Wildlife Services, Environment Canada, 100 Gamelin Blvd., Hull, Quebec, Canada. ** To whom correspondence should be addressed. Abbreviation: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid.

accumulated and excreted [6-11]. With regard to the transport of Mn in blood, the concentration of Mn in normal serum or plasma is several-fold lower than that in erythrocytes (0.5-1.5 # g / l vs. 15-25/~g/1, respectively) [12]. In red cells, it is associated with the hemolysate [13], probably in the form of a Mn-porphyrin complex [14]. However, there is disagreement in the published literature concerning the nature of Mn-binding ligands in serum and plasma, where it has variously been reported to bind primarily to albumin [15], transferrin [16] and 'transmanganin' [17]. Thus, the following series of experiments was undertaken in order to investigate further the binding of Mn and other heavy metals in plasma.

0304-4165/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

164 Materials and Methods

Materials Carrier-free radioactive isotopes 54Mn2+ 65Zn2+, 1°9Cd2+ and 59Fe3+ were obtained from New England Nuclear Corp. (Lachine, Quebec). Nonradioactive manganese (MnC12 • 4 H 2 0 ), zinc (Zn(CH3COO)2.2H20), iron (FeC1 a • 4H20) and copper (CuSO 4- 5H20) were from Fisher Scientific Co. (Fair Lawn, N J). Purified albumin (rat and human) and transferrin (human, 90% iron free) were obtained from Sigma (St. Louis, MO). Sephadex G-75 was purchased from Pharmacia (Dorval, P.Q.), and reagents for polyacrylamide gel electrophoresis were from Bio-Rad Laboratories (Misissauga, Ontario). Blood was collected in heparinized syringes from the median cubital vein of normal, healthy human volunteers, and from the abdominal aorta of anaesthetized rats. Plasma was prepared by centrifugation at 1000 × g for 10 rain. Binding of 54Mn in human and rat plasma Aliquots of plasma (generally 0.2 ml) were incubated in the presence of carrier-free 54Mn2+ (0.1 /LCi) with or without preincubation in the presence of excesses of other metals (Fe, Zn, Cu, 5SMn) for 0.5 h at room temperature. Incubations were carried out in Tris buffer (pH 8.6), after which the incubates were applied on Sephadex G-75 columns (0.9 × 60 cm) and eluted with 10 mM Tris-HCl (pH 8.6) at a flow rate of 20 m l / h . 1-ml fractions were collected and counted for gamma radiation, and the total metal content of the fractions was estimated by atomic absorption spectrophotometry. Binding of 54Mn and other heavy metals to albumin and transferrin Typically, 5 mg of albumin or transferrin were dissolved in 0.2 ml of 10 mM Hepes buffer (pH 7.4), or 10 mM Tris-HCl (pH 8.6) and incubated in the presence of 54Mn2+, 59Fe3+, 1°9Cd2+ or 65Zn2+ (30000-40000 cpm) for 0.5 h at room temperature. The samples were then fractionated on Sephadex G-75 gel filtration columns (0.9 x 60 cm) eluted with either 10 mM Hepes (pH 7.4) or

10 mM Tris (pH 8.6). 1-ml fractions were collected and counted on an LKB model 1270 gamma counter.

Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis was performed essentially according to the guidelines of Davis [18]. Aliquots of plasma (typically 20 /~1) were incubated in 20 mM Tris (pH 7.4) for varying time periods at 37°C in the presence of 54Mn2+ (0.1 p,Ci) a n d / o r other metal cations, then applied on 7.5% polyacrylamide gels (15 cm in length). Electrophoresis was performed at room temperature with a current of 3 mA per tube. The reservoir buffer was 10 mM Tris-glycine (pH 8.3). In order to localize radioactivity, the cylindrical gels were cut into 2-mm sections with a gel slicer, and the radioactivity of each fraction was measured with a gamma counter. Results

Binding of ~4Mn to human and rat plasma Incubation of human or rat plasma with 54Mn2+ resulted in the rapid association of 54Mn with high-molecular-weight proteins as revealed by subsequent G-75 gel filtration (data not shown). Dialysis of the samples before application onto the column did not result in a lower recovery of radioactivity associated with the high molecular weight protein fractions. Preincubation of the plasma samples in the presence of an excess of ~SMn, however, greatly reduced the 54Mn radioactivity subsequently bound to plasma proteins. However, preincubation of aliquots of human plasma in the presence of higher-than-saturating amounts of Fe, Zn and Cu did not eliminate or reduce significantly the binding of 54Mn to the high molecular weight protein fractions. Binding of 54Mn, ~gFe, 65Zn and 1°9Cd to albumin and transferrin Compared to the other heavy metal cations tested in the present study (1°9Cd2+, 65Zn2+, ~Fe3+), 54Mn2+ was found to bind very poorly to purified albumin in aqueous solution at neutral (7.4) or basic (8.6)pH (Fig. 1). The addition of 6 mg exogenous albumin to a 100 t~l aliquot of rat plasma did not result in an increased level of 54Mn

165 Albumin

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of fractions from Sephadex G-75 fractionation of 5 mg albumin preincubated in the presence of 0.2 #Ci carrier free 6SZn2+ for 0.5 h, then 1 mM Mn2+ for an additional 0.5 h. Mn was unable to displace 65Zn from the protein. Elution buffer, 10 mM Hepes (pH 7.4).

spite the fact that the protein content was more than doubled due to the added albumin. Also, the incubation of 65Zn-albumin in the presence of excess Mn 2+ (1 mM) did not result in the displacement of 65Zn from the protein (Fig. 2). In contrast to the results with albumin, 54Mn became associated with transferrin, not as well as 59Fe3+, but considerably better than 65Zn2+ or 1°9Cd2+ under the conditions of the present study (Fig. 3). Binding was more complete at a basic pH than under neutral conditions. Preincubation of transferrin in the presence of just sufficient Fe to saturate the specific binding sites (1.6 #g F e / m g protein) completely prevented the binding of subsequently added 54Mn2+ (Fig. 4), indicating that Mn probably binds to the Fe sites on the protein if the protein is not saturated with Fe.

Polyacrylamide gel electrophoresis of human and rat plasma Because of the inability of Sephadex G-75 gel filtration to adequately distinguish among plasma proteins, electrophoresis of plasma samples incubated with 54Mn2+ was attempted. Fig. 5 shows the excellent degree of protein separatiod that can be achieved by this method. Incubation of either human or rat plasma with 54Mn2+ for 24 h at 37°C resulted in the association of 54Mn and 5~Fe with a protein having the electrophoretic mobility of transferrin (Fig. 6). A second, less prominent peak of 54Mn radioactivity was associated with electrophoretically slow ligands which remained in the stacking gel or just barely entered separating gel. In order to eliminate the possibility that 54Mn was binding not to transferrin but to another protein with a similar mobility, a plasma sample was preincubated for 15 min at room temperature with enough Fe z+ to cause saturation of transferrin (2 /.tg F e 2 + / 2 0 ~1 plasma) before incubation with 54Mn2+. This resulted in a complete absence of 54Mn radioactivity associated with the transferrin region and a corresponding increase in the 54Mn associated with the electrophoretically slow ligands (Fig. 7). Preincubation in the presence of a similar concentration of Zn 2 +, however, resulted in an electrophoretic pattern of recovered radioactivity similar to that seen in the absence of any competing metals. The specific effect of Fe in preventing the binding of 54Mn indicates that it is

166

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mainly transferrin with which Mn associates, provided that Fe-binding sites are not saturated. The binding of 54Mn to plasma transferrin was dependent upon incubation time (Fig. 8). When no incubation time was allowed, 40-60% of the applied radioactivity was recovered from the gel, and of this recovered radioactivity, less than 10% was associated with transferrin; 50-60% was bound to 'slow' ligands (those remaining in the stacking gel plus the first 4 m m of the separating gel). After 0.5 h incubation, virtually all ( > 90%) of the total applied radioactivity could be recovered from the gel. At this time, most of the 54Mn was still associated with slow ligands, but progressively more radioactivity became bound to transferrin as incubation time was increased reaching a maximum between 5 and 12 h. There was never more than 5% of the recovered radioactivity associated with albumin. When a solution of 54Mn2 ~ alone, or 54Mn2 ' in 10 mM histidine, was applied on the gel, no radio-

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activity was recovered from the stacking gel or from the separating gel. Discussion There has been considerable disagreement in the published literature concerning the nature of Mn-binding ligands in serum and plasma. Foradori et al. [19] reported that approx. 80% of 54Mn added to plasma in vitro became bound to a /3cglobulin, and Cotzias and Bertinchamps [17]

II

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Fig. 6. Electrophoretic pattern of 54Mn ( ) and 59Fe (----) radioactivity in human or rat plasma after 24 h incubation at 37°C, pH 7.4, in the presence of cartier-free 54Mn2+ or 59Fe3+. SG, stacking gel; Tf, transferrin; Ab, albumin; DF, dye front.

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Fig. 7. Electrophoretic pattern of 54Mn radioactivity in 20 ffl human plasma incubated for 24 h at 37°C, pH 7.4, in the presence of 0.1 ,ttCi 54Mn2+, @, 54Mn2+ alone; or preincubated for 15 rain at room temperature with 2.0 #g Fe 2. (O) or Zn ~+ (D). SG, stacking gel; Tf, transferrin; DF, dye front.

168 had previously stated that this ligand was not transferrin since the protein-bound 54Mn did not exchange with Fe. These researchers hypothesized the existence of a specific Mn-transport protein in plasma (transmanganin). Later, Keefer et al. [16] reported that, in rat serum, a single protein binds the majority of both 54Mn and 59Fe. The isoelectric point of the protein was determined to be 5.7, which is close to that of human transferrin (5.9). It was concluded that 54Mn binds primarily to transferrin in rat serum. However, Nandedkar et al, [15] suggested that Mn combines selectively with albumin in human and rabbit plasma. This conclusion was based largely on the finding that when 54MNC12 was mixed with serum in vitro and then applied on a Sephadex G-100 column, the emergence of radioactivity coincided with that of albumin. But albumin, transferrin and other proteins of similar molecular mass cannot be efficiently separated on a Sephadex G-100 column. There is no doubt that Mn 2+ can bind to albumin [20], but this does not mean that, in the presence of many other potential ligands, Mn will bind preferentially to albumin. In the present study, we have shown that Mn binds very poorly to purified albumin compared with other divalent heavy metal cations (Figs. 1 and 2). The binding of Mn to albumin, in our view, cannot possibly account for more than a very minor portion of the total Mn bound to plasma proteins. On the other hand, Mn binds purified transferrin better than Zn or Cd (Fig. 3) probably occupying Fe binding sites (Fig. 4). In addition, the present study also demonstrates the influence of time (Fig. 6) and pH (Fig. 2) on the association of 54Mn with transferrin. Aisen et al, [21] have shown that Mn, when complexed with transferrin, is present exclusively in the trivalent state. At neutral and acidic pH values, Mn 2+ is extremely stable, but basic pH values provide a more favorable condition for the oxidation of Mn 2 + to Mn 3 +. In plasma, oxidizing agents such as ceruloplasmin may play a role in converting divalent Mn to the trivalent form [23]. Thus, given enough time, Mn 2 + will be oxidized to Mn 3+ and will become selectively bound to transferrin in both human and rat plasma (Figs. 6 and 7). The major binding ligand for divalent Mn, however, cannot be transferrin. This is illlustrated in Fig. 8 where it can be seen that, when insufficient time

has been given for the oxidation of 54Mn2' tO 54Mn3+, most of the recovered radioactivity is associated with electrophoretically slow plasma components, That there are Mn-binding ligands in plasma other than transferrin is also illustrated by the fact that plasma which has been saturated with Fe (as well as Zn and Cu) can still bind 54Mn (data not shown), and by the results of Lau and Sarkar [22], who found that, when 54Mn2+ was mixed with human serum and allowed to equilibrate overnight at 4°C, a major portion of the recovered radioactivity after Sephadex G-100 filtration was associated with ligands of higher molecular mass than serum albumin. A possible candidate for this ligand is %-macroglobulin which, when isolated, has been shown to bind 54Mn [23], as well as other metals such as Zn [24]. However, when plasma that has been incubated with 54Mn is subjected to zone electrophoresis, the majority of non-/~-globulin bound radioactivity migrates with the y-globulins not the c~-globulins [19]. Further investigations are thus required to better characterize the high molecular weight, electrophoretically slow Mn-binding component(s) of plasma. Data presented in the present study are suggestive of a critical role for the valence state of Mn with regard to the metal's plasma-binding characteristics. This has relevance to the pharmacology and toxicology of Mn. Numerous investigations on experimental animals have been carried out over the last 20 years in an attempt to understand bettter how Mn is distributed, excreted and accumulated inorganisms. Often these studies have involved the intravenous or intraperitoneal injection of soluble divalent Mn salts [6,8,10,13,25,26] and have resulted in the finding that injected Mn e+ is cleared very rapidly from the blood and efficiently secreted into the bile. It thus has a short biological half-life compared with other metals. Chronic administration is required in order to observe any substantial increase in the Mn content of tissues. On the basis of the present study, however, it might be expected that trivalent Mn which, unlike divalent Mn, binds avidly to transferrin, might reveal a significantly different profile of distribution, excretion and accumulation than that observed for Mn 2 +. Initial support for this idea comes from Gibbons et al. [23], who found that the

169

plasma clearance of free 54Mn2+ after portal administration to cows was about 30-times more rapid than that of 54Mn complexed with transferrin. illustrating that once Mn 2+ is converted to Mn 3+, it is less efficiently cleared and excreted by the liver. Also, regarding the toxicity of Mn, Mn 3+ (but not Mn 2+) is a strong oxidant. It was Rodier [27] who first proposed that, in the case of occupationally exposed miners, the most neurotoxic of the Mn ores were those containing the metal in a valence state higher than 2. Thus Mn 3+ not only has a longer biological half-life than Mn 2+, it is probably also the more toxic of the two forms. Future investigations on the toxicokinetics of Mn should recognize the critical importance of the valence state of Mn, particularly the possibility that, unlike Mn 2+ which is rapidly and efficiently excreted by the liver, there may be a tendency for Mn 3+ to be accumulated by tissues. Much of the Mn which is not initially excreted in the bile is probably, like Fe, delivered to neurons and other cells primarily as Mn 3+-transferrin complex.

Acknowledgement The authors wish to thank Mrs. Colleen Alford for secretarial assistance.

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