Selenium in human plasma: Levels in blood proteins and behavior upon dialysis, acidification, and reduction

BIOINORGANIC

Selenium

CHEMISTRY

in Human

3,217~223

Plasma:

Proteins and Behavior upon Acidifkation, and Reduction

(1974)

Levels

217

in Blood

Dialysis,

W. J. RHEAD , G. A. EVANS, and G. N. SCHRAUZER Department of Chemistry, Unicersily of Colifomia, San Diego, &wll.~ Cdlege, La Jo&z, California 92057 ABSTRACT A number of human blood proteins and other proteins were analyzed for selenium. as was whole human plasma after dialysis under various conditions; the proportions of acid-volatile and zinc-HCl reducible selenium in plasma were aLsodetermined. Excluding ceruloplasminand the QIglobulins, which have elevated selenium contents, the selenium levels of most proteins ap+ar to parallel their sulfur contents. Only 30% of native plasma selenium can be mobilized by diiysis against reducing or chelating agents or reduced to H&e by vigorous reduction with Zn-HCl; our results are compared to those of other studies employing %eC~‘. Contrary to earlier reports, selenate is reduced to H&z by 2%HCl, indicating that this method of reduction is not specific for selenite.

KEY

WORDS

Selenium; human plasma; protein levels; chemical behavior

INTRODUCTION The native selenium in plasma and tissues is present in dialysable and undialysable forms [l-5]. The dialysable fraction is presumably present in the form of reducibleselenotrisul6des(RSSe-SR) [2,6,7], but also contains selenium in other oxidation states, e.g., in the form of protein-bound selenide (SeA2), selenite (Se*), or even selenate (Se*), [S] as well 8s organically bound selenium [9]. The presence of selenium in merent oxidation states in tim was first suggestedby observationson the behavior of selenium-75 in tissue fractions [8]: A portion of the total selenium-75 volatilizedon acidificationwith HCl, indicatingthe presenceof selenide.An additional amount of selenium-75 could be volaGlimd on reduction with @ American ELsevierPublishing Company, Inc., 1974

2l8

W. J. RHEAD ET AL.

metallic Zn-HCl and was for this reason considered to be selenite, although sel&ate is also reduced under these conditions [IO]. Animal studies [l-3] indicated that serum globulins bind and retain a large proportion of but similar studies have apparently not been administered ‘sseo,-, pedormed with human blood proteins. Furthermore, no data are available on the distribution of dialysable and undialysable reduced or oxidized forms of native selenium in human plasma [ll]. In view of the afEnity of selenium for sulfur and their chemical similarities, sulfur-containing native proteins may be expected to contain traces of selenium in amounts approzimately proportional to their total sulfur content, although the selenium content of speeifk selenium transport proteins OF of selenium-dependent enzymes (viz. glutathione peroxidase) [l2] could be disproportionately higher. To obtain some initial information, we have analysed 11 human blood proteins and 7 other purified proteins for seienium. We have also determined the amounts of dialysable and undialysable native selenium in human plasma, as well az the proportions of acid-volatile and Zn-HCl reducible selenium. -. MATERIALS Samples and Elemental

AND METHODS

Anal+s

Normal human blood proteins were obtained from Mann Research Laboratories, New York, N-Y.

10006 and the other proteins from CaJbio-

them, San Diego,. Calif. 92937 and Nutritional Biochemicals Corp., Cleveland, Ohio 44128. Upon receipt in our laboratory, all proteins E-ereanalyzed for selenium xithout further puScation. Pooled normal human plasma samples free from hemolysis were obtained from San Diego Blood Bank, San Diego, California or from volunteers in our laboratory. Total selenium content of our samples was determined by a simple modification [13] of t.he guorometric method of Olson [14] ; all reagents were the purest commercially available (USP or reagent grade). The sulfur contents of the proteins were calculated from their methionine and cysteine contents [E] or determined by Schwarzkopf Microanalytical Laboratory, Woodside, N. Y. 11377. Reductive Dklysis

Experiment

Duplicate 3-ml samples of pooled human plasma were dialysed for 72 h at 25°C against 3.5 1 of 0.15 M Tris-HCl buffer, pH = 7.4 with or xvithout added reducing or chelating agents (lo+ M; Table 1). The buffer solutions

were deaerated with NZ gas before adding the reducing agents or plasma

SELE?XUM

IN HUMAN TABLE

219

PLASMA

1

Effect of Dialysis and Various Agents on the Selenium Content of Human Plasma

Dialysis Solution

Selenium Content of Plasma (ppm)

% of Control

None None

0 _17 (Control) 0.16

100 94

Cysteine SOSGlutatbione SEDTA

0.16 0.16 0.14 0.12 0.12

Agent (10-W)

h’one 0.15 32 Tris Buffer pH-7.4 I,

II ,, II I,

94 94 82 70 70

samples, and N= gas was passed through the solutions during the entire experiment_ At the end of 72 h, the dialysed samples, free from visible precipitation, were analy&d for total selenium content. Oxidation

State

Experiment

The method of Diplock et al. [S] was followed as closely as possible. Either 1 ml of cont. HCI or 1 ml of cone. HCI t 200 mg Zn dust was added to duplicate l-ml samples of fresh human plasma at 25°C; i\l’ngas was then passed through the sohrtions for 15 min and the samples were assayed for total selenium (Table 2). Duplicate l-ml samples of SeOr solutions in d&i&d H*O (0.1 ppm) were carried through the entire procedure in parallel to the plasma samples. The HCI-volatile selenium is considered to be “selenide,” the HCI-Zn reducible selenium ‘belenite,” and the remainder %elenate” and/or organic TABLE

2

Reduction of Selenium in Human Plasma to H&k by HCI-Xt and HCl-Zn-h=_ % Total Selenium Reduced to H&e f SampIe Treatment None (Control) HCI + X2 HCL f Zn f N+

Plasma o9*2 29 I 6

SD. (N = 6)

Se02 Std. oOf1 92 I 4

W. J. RBEXD

220

ET AL.

selenium compounds [S]. However, both examination of the standard reduction potentials [lo] and experiments in our laboratory demonstrate that Zn-EC1 reduces SeO,- to H&e under our experimental conditions; thus, only organ*selenium compounds are resistant to reduction by Zn-HCl. Also, n-hen deter-mink g the proportion of acid-volatile selenium, the & gas must be passed through the HCI-plasma mixture via a glass pipette; metal syringe needles presumably contain zinc and tin which reduce 30 to 50% of a Se02 standard to HzSe upon contact with 50yo HCI, thus elevating the observed proportion of “selenide.” The apparent acid lability of the Se02 standards decreases dramatically when glass pipettes are substituted for metal needles. RESULTS Selenium

Content

of Proteins

The selenium levels of proteins from human blood and other sources are shown in Table 3. While our values for the native selenium content of the plasma proteins agree with those determined by neutron activation analysis, we found more selenium in human albumin than did Dickson and Tomliin [ll]_However, their albumin sample was obtained from a different source and dialysed estensively against deionized water. The selenium levels cf the first 17 proteins in Table 1 show little correlation with their sulfur contents, i.e., the sum of sulfur in cysteine and methionine (r = 0.354, p > 0.03) [15]_ However, the &Oglobulins and ceruloplasmin contain more selenium than do the other proteins; it has also been reported that the ~2 globulins, which include ceruloplasmin, [15] preferentially bind %e03in LIIvo,[3] and thus might have specific roles in selenium metabolism. Excluding these two proteins from consideration, the selenium contents of the remaining 15 proteins paralIe1 their sulfur contents quite well (r = 0.830, p < 0.01); approximately one selenium for sulfur substitution per 25,000 sulfur atoms would account for the observed selenium levels. Glutathione, a tripeptide, (%S = 10.21) has a disproportionately low selenium content (1.04 ppm), since seleno-glutathione is probably separated from glutathione during purification due to its different chemical properties (increased ease of osidation, etc.). Behavior of Plasma Reduction

Selenium

Upon

Dialysis,

Acidification,

and

The fraction of native selenium removed from plasma by anaerobic dialysis in the presence of various agents is shown in Table 1. Approximately

SELENIUM

IN HUMAN

PLASMA

221

TABLE 3 Selenium Content of Proteins

Human Blood Fractions Hemoglobin (lOOo/,) Globin Fibrinogen (Cohn-Fraction I-65% clottable) 75 y Globulin (100%) y Globulins (Cohn Fraction II-SS%,) ~2 Globulins (70%) aI Globulins (Cohn Fraction IV) Albumin (100%) Glycoprotein (Cohn Fraction VI)

Transferrin (95 %) Ceruloplasmin

Selenium Content (ppm Dry Weight) 0.65 0.76 1.70 0.85 0.65

$&%lfur (N-t/W) calculated (15) 0.498

0.517 1.122

3.37 5.42”

0.712 0.5Ob 0.72b 0.75b l-S62 0.820 1.912 0.710

0.15 1.39

1.114 0.961

2.01

o.s33

0.67

0.95

4.05 O-M 1.91

3.356 0.176 (not determined)

5.16 1.3i 1.15 0.96

Other Proteins: a-Axnylase (B. subtilis) Pepsin (Bovine) Catalase (Bovine) Hexokinase (C_ albiuw~) Insulin (Bovine) Collagen (Bovine) Dioldehydrase (A. aerogenes) Other Blood Components: Glutathione Meraptalbumin Stroms

1.04 1.4i 2.9s

10.21 1.862 0.866

o Corrected for fluorescence due to copper. bDetermined by Schwarzkopf MicroanaIyticnI Laboratory, Xew York, 3i.Y. 113'77.

6% of the plasma selenium n-as removed by diaIysis against buffer, sulfite ion, or cysteine. Reduced glutathione and sulfide ion are more effective, mobiimg 18 and 30% of the total plasma selenium, respectively. The large proportion of plasma selenium released by EDTA suggests that metal ions may participate in selenium binding to plasma proteins_ The proportions of selenium in plasma and Se02 standards volatilized by HCl-X2 and HCl-Zn& are shown in Table 2. Oniy 10% of plasma selenium

222

IV_ J. RHEXD

ET AL.

is acid-volatile and is probably present as seienide; 30% of plasma selenium is removed by Zn-HCl, suggesting that 20% of the selenium in our plasma samples is in the form of seienite snd/or seienate. Over 60% of native plasma selenium is not accessible to either reducing or chelating agents (Table 1) or vigorous reduction (Table 2) and thus is most likely in the form of organically bound selenium

DISCUSSION This study suggests that the selenium levels of proteins may correlate with their sulfur content in cysteine and methionine (Table 3), as do the selenium levels of crop plants grown on selenium-supplemented soil with their sulfur contents [16]. Traces of selenium may also be present in the form of seleno-methionine, n-hi& can replace methionine in the alga Ci&welZuand in methionine-requiring mutants of E. coli [9]. In the rat, 75Se-Lseien*methionine is significant-iy incorporated into plasma crfhaptoglobin and -macrogiobulin following turpentine injury [17]. Another fraction of selenium may also be present in seienotrisuifides (RS-Se-SR), formed in proteins in r&o and possibiy in tGo [2, 6, 71. Thus, several mechanisms suggest that the selenium levels of proteins would parallel their content in t.he sulfur amino acids. Other studies have suggested &at the ap or y-globulins of plasma bind and transport selenium [l, 31; in support of some of these studies, we found that cerulopiasmin, an aTglobulin, and the cwglobulins have unaccountabIy high seIenium 1eveI.s. Only 30yo of the total plasma selenium is released by dialysis against 1CP3~11reducing and chelating agents (Table 1).In contrast, after aclmin&ration of YSeOs- to animals, approximately 70-90~o of the%e complexed with chick plasma proteins [2] x-as released by dialysis against thiols, sulfite, or dilute NaOH, as was 50-75Q/, of the We in rat bile and liver homogenates L-4, 51. Furthermore, less than 10% of the native human plasma selenium is acid volatile (Se-‘) and only 207, is Zn-HCi reducible, (Se*4, Se*; Table 2). In the liver cell fractions of rats given YYeOj- orally, Dipiock et al. [S] found more acid volatile We (6-50%) and similar proportions of zinc-acid reducible ‘%e (20-5070). Although seienate as well as selenite can be reduced to H&e by Zn-HCl in z&o [lo] (see Materials and Methods), Dipiock noted that a certain proportion of the %e always resisted reduction, [81 suggesting its rapid conversion to non-volatile organoseienium compounds. Monogastric mammals can not directly incorporate seienite into cysteine or methionine [9]; however, the bacteria of the intestinal tract (Le., E. co&_) can synthesize organoselenium compounds from iSSeOJ-, [18] which may explain the conversion of ‘SSe to

SELENIUM

IN HUMAN

PLASMA

223

non4abiie reduced forms in ~~tirm.Our experiments indicate that 70% of native plasma selenium is not accessibIe to either dialysis or reduction and appears to be either firmly bound to or incorporated into proteink, in contrast to the resulti of animal studies employing 7sSf203-. This work teas suppmied by grads GP &&$58X of the National Science Foundation, and l-R01

CA l%V64-OlAl of ihe Nationd

ImtiLute

of

Health.

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