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ORIGINAL PAPER

Selenium speciation in paired serum and cerebrospinal

fluid samples

Nikolay Solovyev & Achim Berthele & Bernhard Michalke

Received: 21 May 2012 /Revised: 18 July 2012 / Accepted: 20 July 2012 /Published online: 7 August 2012# Springer-Verlag 2012

Abstract Se speciation was performed in 24 individual

paired serum and cerebrospinal fluid (CSF) samples from

neurologically healthy persons. Strong anion exchange(SAX) separation, coupled to inductively coupled plasma –

dynamic reaction cell – mass spectrometry (ICP-DRC-MS),

was employed. Species identification was done by standard

matched retention time, standard addition and by size ex-

clusion chromatography followed from SAX (2-D SEC-

SAX-ICP-DRC-MS) and by SAX followed from CE-ICP-

DRC-MS (2-D SAX-CE-ICP-DRC-MS). Limit of detection

(LoD, 3×standard deviation (SD) of noise) was in the range

of 0.026 – 0.031 μ g/L for all investigated species and thus

was set uniformly to 0.032 μ g/L. Quality control for total Se

determination was performed by analysing control materials

“human serum” and “urine”, where determined values met target values. Several Se species were found in both sample

types having following median values (sequence: serum/

CSF, each in μ g Se/L): total Se, 58.39/0.86; selenoprotein

P (SePP), 5.19/0.47; Se-methionine (SeM), 0.23/<LoD; glu-

tathione peroxidase (GPx), 4.2/0.036; thioredoxinreductase

(TrxR), 1.64/0.035; Se IV, 12.25/0.046; Se-human serum

albumin (Se-HSA), 18.03/0.068. Other Se species, such as

Se-cystine (SeC), Se VI and up to four non-identified compounds were monitored (if ever) only in very few samples

usually close to LoD. Therefore, their median values were

<LoD. Linear relationships based on median values provide

information about Se-species passage across neural barriers

(NB): SePP-serum is significantly correlated to total Se-serumwhen the latter was >65 μ g/L; however, SePP-CSF appeared

independent of SePP-serum. For Se-HSA-serum versus (vs.)

Se-HSA-CSF, a weak linear relationship was found (r 20

0.1722). On the contrary, for anti-oxidative Se-enzymes,

higher r 2 values were calculated: GPx-serum vs. GPx-CSF,

r 200.3837; TrxR -serum vs. TrxR -CSF, r 200.6293. Q-Se-species

values (0

ratios of CSF-Se-species/serum-Se-species) were compared with the Q-Alb value (HSA-CSF/HSA-serum0clinical

index of NB integrity) for deeper information about NB

passage of Se species. The Q-Se-HSA value (3.8×10−3) was

in accordance to the molecular mass dependent restriction at

NB (Q-Alb at 5.25×10−3). Increased Q values were seen for

TrxR (21.3×10−3) and GPx (8.3×10−3) which are not

(completely) explained by molecular size. For these two

anti-oxidative Se-enzymes (GPx, TrxR), we hypothesize

that there might be either a facilitated diffusion across NB

or they might be additionally synthesized in the brain.

Keywords Selenium speciation . Cerebrospinal fluid .Serum . Thioredoxin reductase . Glutathione peroxidase

Introduction

Selenium is an essential trace element for humans. Its ben-

eficial role for human health is generally accepted and

includes protection against oxidative stress, prevention of

heart diseases and cancer or optimal immune responses

Published in the topical collection Metallomics with guest editors Uwe

Karst and Michael Sperling.

N. Solovyev

Institute of Toxicology, FMBA,

St. Petersburg 192019, Russia

A. Berthele

Department of Neurology, Klinikum rechts der Isar,

Technische Universität München,

81675 Munich, Germany

B. Michalke (*)

Research Unit Analytical BioGeoChemistry,

Helmholtz Center Munich — German

Research Center for Environmental Health,

Ingolstädter Landstr. 1,

85764 Neuherberg, Germany

e-mail: [email protected]

Anal Bioanal Chem (2013) 405:1875 – 1884

DOI 10.1007/s00216-012-6294-y

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[1 – 6]. Innate and adaptive immunity are impaired in Se-

deficient individuals. Increased Se intake boosted Ca 2+ flux

and downstream cell signaling in CD4+ T cells, which

influenced their activation, proliferation and differentiation

[7]. The biological effects of Se are exerted mainly through

its incorporation into selenoproteins. There, Se is incorpo-

rated as SeC where it has been shown to inhibit NF-kappa B

activation, which leads to reduced expression of NF-kappa B target genes, including inflammatory cytokines [8]. Thy-

roid metabolism, too, is dependent on Se as deiodinases are

selenoproteins. Some selenoproteins, such as GPx and

TrxR, have been functionally characterized as antioxidant

enzymes, serving to mitigate damage caused by reactive

oxygen species (ROS) and to regulate redox tone [9]. Both

GPx and TrxR are present in brain [ 10]. In the central

nervous system, oxidative stress has been implicated in

pathophysiology of neurodegenerative diseas es, such as

Alzheimer ’s (AD) and Parkinson’s disease (PD) and even

stroke [10 – 12]. Fahn and Cohen [13] published data indi-

cating oxidative stress specifically in the substantia nigra of PD patients. Brain Se levels are primarily maintained by

selenoprotein P (SePP) directly expressed in neuronal tissue

[14], which in turn depends on proper Se supply across the

blood – brain barrier (BBB). Cerebrospinal fluid (CSF) is

mainly an excretion of the choroid plexus in the brain

ventricles. It plays an important role in the homeostasis

and metabolism of the central nervous system. Since CSF

has close molecular exchange with the extracellular space of

brain parenchyma, the composition of CSF and extracellular

brain fluids are similar and a misbalance, a depletion of

elements or change of element species in the brain is likely

to be reflected in CSF, which is summarized by Siverling

[15]: “Cerebrospinal fluid is a vital matrix for understanding

the events ongoing in the brain as the medium bathes the

brain, yet does not come into contact with other organs or

fluid compartments under normal physiological conditions,

thereby producing highly dependable information

concerning brain pathology”. Therefore, Se depletion or

changes in Se speciation in neurological diseases can be

monitored in the CSF. Total Se concentration in CSF was

described to be low in healthy control subjects (approxi-

mately 1.2 – 4 μ g/L) in recent papers which adhere to suffi-

cient quality control (QC) measures [16 – 18].

The transport mode of Se species to the brain across NB

is not completely understood. In previous work, we have

shown that the total Se concentration in CSF is independent

from serum Se concentration and a strictly controlled, reg-

ulated pathway across NB was assumed [17]. Se species in

CSF were investigated where four Se compounds were

identified but several remained unknown [18]. A promising

concept to understand, in which form Se is crossing the NB,

is the analysis of paired serum (before NB) and CSF sam-

ples (behind NB). No Se speciation data of such paired

samples are published to the best of our knowledge, except

for SePP. In mice, SePP was found to be transcribed in brain

independently from liver-borne SePP [14].

In the present paper, the analysis of Se species in paired

serum and CSF of 24 individuals (a ’ 2 replicates) was

performed with SAX-ICP-DRC-MS. The performance for

species identification was 2D by SEC-SAX- and SAX-CE-

ICP-DRC-MS. The DRC mode of the mass spectrometer was chosen for removing interferences on Se isotopes. A

previously developed SAX method from Xu et al. [19] was

modified and used for separation of SePP, SeM, GPx, SeC,

TrxR, Se-HSA, selenite and selenate.

Experimental

Chemicals and reagents

Chemicals and reagents used throughout this work were of

suprapure grade. The chemical list consists of the following:certified selenium and rhodium stock standards (1,000 mg/L,

CPI, Santa Rosa, USA); selenite, selenate, SeM, SeC, TrxR

(EC 1.8.1.9.), GPx (EC 232-749-6), human serum albumin

(HSA) and TRIS buffer (Sigma-Aldrich, Deisenhofen, Ger-

many); protein standards for mass calibration of a size exclu-

sion column were γ-globuline (150 kDa), arginase (107 kDa),

glutathione peroxidise (86 kDa) transferrin (78 kDa), albumin

(66.5 kDa),β-lactoglobuline (36.5 kDa), lysozyme (14.3 kDa),

metallothionein (7 kDa), each from Sigma-Aldrich; ammoni-

um acetate and acetic acid (Merck, Darmstadt, Germany); Ar liqand methane (99.999 % purity, Air Liquide, Gröbenzell,

Germany).

Samples

Serum and CSF sample pairs, drawn at the Department of

Neurology of the Technische Universität München, were

obtained from 24 patients with unspecific neurological com-

plaints, like headache, dizziness and various sensory symp-

toms. CSF and serum samples were collected and handled as

described previously [18]. In short terms, CSF was collected

from each individual by standardized lumbar puncture and

serum was obtained from blood drawn from the cubital vein

directly after the spinal tab. Thus CSF andserum are referred to

as “ paired samples”. In the case of unremarkable CSF test

results, CSF and serum samples were considered to origin

from neurologically healthy individuals. After patients con-

sented to the use of their samples for scientific investigations,

the previously aliquoted, frozen-stored samples were subjected

to total Se determination by FI-ICP-DRC-MS [17] and subse-

quently to Se speciation. The samples were thawed at 4 °C in

the refrigerator, vortexed (and only for serum samples: diluted

1/5 with Milli-Q water) and injected to SAX-ICP-DRC-MS.

1876 N. Solovyev et al.

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Selenite and selenate stock solutions were prepared at a

concentration of 1,000 mg Se/L by dissolving in Milli-Q

water (18.2 MΩ cm, Milli-Q system, Millipore, Bedford,

MA, USA). HSA was prepared accordingly at a concentra-

tion of 1,000 mg/L. Preparation of an Se-HSA was done by

mixing 10 mg Se/L selenite to this stock solution and

incubation for at least 14 days. Working standards of Se

species were prepared daily from their stock standard sol-utions by appropriate dilution with Milli-Q water.

HPLC parameters

Strong anion exchange (SAX)

SAX separation was based on the method of Xu et al. [ 19],

but slightly modified, to ensure baseline separation of close

eluting peaks mainly in the middle of the chromatographic

gradient [18]. A Knauer 1100 Smartline inert Series gradient

HPLC system was connected to an anion exchange column

ProPac SAX-10 (250× 2 mm ID) from Dionex (Idstein,Germany). The sample volume was 100 μ L. The mobile

phases were: eluent A, 10 mM Tris – HAc buffer, pH 8.0; and

eluent B, A+500 mM ammonium acetate, pH 8.0. Gradient

elution was as follows: 0 – 3 min 100 % eluent A; 3 – 10 min

100 – 60 % eluent A (0 – 40 % eluent B); 10 – 23 min 60 – 45 %

eluent A (40 – 55 % eluent B); 23 – 26 min 45 – 43 % eluent A

(55 – 57 % eluent B); 26 – 28 min 43 – 0 % eluent A (57 –

100 % eluent B); 28 – 52 min 100 % eluent B, 52 – 60 min

100 % eluent A. The flow rate was 0.20 mL min−1. The

column effluent was mixed with 1 μ g/L Rh (final concen-

tration) as internal standard (total flow rate: 0.25 mL min−1)

and directed to ICP-MS.

Size exclusion chromatography (SEC)

SEC was used to prepare mass range-characterized SEC

fractions for a 2D approach for species identification. The

Knauer 1100 Smartline inert HPLC system was connected

to a Biobasic 300mesh column (300×7.8 mm ID, Fischer

Scientific, separation range ca. 800−3 kDa). Tris – HAc

(10 mM, pH 7.4) +250 mM NH4Ac was used as the eluent

at a flow rate of 0.75 mL min−1. Mass calibration was

performed using protein standards of defined molecular

mass. Retention times related to molecular masses followed

the equation “ln(Da)0−263×RT+13.597” (r ²00.9916). For

subsequent analysis by the regular SAX-ICP-MS method,

the SEC column effluent was fractionated by a “Fraction

Collector 100” (Pharmacia, Freiburg, Germany). Fractions

were frozen at −20 °C and freeze dried (Heraeus-Christ,

type “Beta ”, Osterode, Germany). The freeze-dried, size-

characterized fractions were dissolved in 300 μ L Milli-Q

water and kept frozen again until SAX-ICP-DRC-MS

measurements.

Capillary electrophoresis (CE)-ICP-DRC-MS

A “Biofocus 3000” capillary electrophoresis system (Bio-

Rad, Munich, Germany), equipped with an uncoated capil-

lary (CS-Chromatographie Service GmbH, Langerwehe,

Germany) 120 cm× 50 μ m ID was used for CE-ICP-DRC-

MS analysis. Hyphenation is detailed in [20]. Before each

run, the capillary was purged with NH4-acetate/acetic acid buffer, 10 mM, pH 3.0 (70 s, 8 bar). Pressurized sample

injection was performed for 2 s, followed from 1 s buffer

injection. The separation voltage was +25 kV. A sheath flow

(diluted running buffer 1/25) around outlet electrode and

capillary end at nebulizer provided the electrical connection.

Preparation of SePP from human serum as a standard

SePP is not commercially available. However, several

papers describe a selective purification from human plasma

using affinity chromatography [21]. This method is used

also by Jitauru et al. for Se-speciation investigations [22].We slightly modified the elution program according to the

column instruction sheet. Briefly, 200 μ L serum was

injected on a Heparin-affinity column (Amersham, GE

Healthcare Europe GmbH, Munich, Germany) and chroma-

tographed by a linear 12-min lasting gradient of A050 mM

Tris, 10 mM NH4-acetate/acetic acid, pH 6.0 to buffer B0A

but 800 mM NH4-acetate, pH 8.5, and remaining at 100 % B

for further 8 min. SePP was eluted at a flow rate of

1.0 mL min−1 and collected under 280 nm monitoring. Se

was subsequently determined in fractions with FI-ICP-

DRC-MS [17]. The SePP fraction was preconcentrated by

freeze drying and was re-dissolved in 1 mL of 10 mM Tris –

HCl buffer, pH 7.2. The resulting SePP solution was imme-

diately frozen and stored at −20 °C until use. For verifica-

tion, an aliquot of this affinity chromatographic SePP

fraction was analysed additionally on the thoroughly mass

calibrated SEC column, where it eluted at RT calculated for

60 kDa, which fits to literature data [23].

Inductively coupled plasma mass spectrometry

Table 1 shows the experimental settings chosen for ICP-

DRC-MS after optimization.

Total Se determination and peak quantification from

chromatograms

Flow injection analysis ICP-DRC-MS was applied for total

Se determination according to our previously published

method [17]. In short terms, a Knauer 1100 Smartline inert

Series binary HPLC system equipped with vacuum degasser

and an electronic valve with a 25-μ L injection loop (Perkin

Elmer, Rodgau-Jügesheim, Germany) was directly coupled

Selenium speciation in paired serum 1877

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to ICP-DRC-MS. The flow rate was 1 mL min−1 of 50 %

eluent A and B, each from SAX separation. CSF samples

were diluted 1:1.1 by adding 1 μ g/L Rh (final conc.) as

internal standard.

Peak quantification from FI-quantification and from

chromatograms was done by comparing peak areas with

peak area calibration curves from FI-ICP-DRC-MS. Stan-

dard addition method was used for standard retention time

matched identification of Se species and as QC means in

quantification.

Data processing of FI-ICP-(DRC)-MS

Rh and Se data files were exported from the NexIon soft-

ware and processed with the Knauer HPLC software “Clar-

ity” for peak area integration. For each sample (or standard),

a quotient of Se-peak area to Rh peak-area was calculated

and taken as the result corrected for the internal standard

(Rh). These values were used for the calibration curve

(standards) or for calculating the concentration according

to the calibration curve (samples).

Quality control experiments

Total Se determination Quality control for total Se deter-

mination was performed by analysing control materials

“human serum” and “urine” from Recipe, Munich. Con-

trol materials were reconstituted as indicated on flask

labels and the resulting solutions were diluted 1/50

(serum) or 1/10 (urine) with Milli-Q water before

measurements.

Mass balances during SAX-ICP-DRC-MS Defined amounts

of single Se species (related to Se: solutions prepared at

10 μ g/L) of those Se species which were regularly above

LoD, were injected to the SAX-ICP-DRC-MS system and

peak Se concentrations were quantified. These Se concen-

trations were related to the injected Se amounts (0100 %)

for recovery calculation. Analogous, serum or CSF samples

were quantified for total Se (0100 %) before injection. Thesum of eluted and quantified peaks was subsequently related

to this total Se concentration.

Species identification Additionally to standard retention time

match and standard addition, species identification was per-

formed by two 2D approaches: (a) mass range defined SEC

fractions were analysed with the regular SAX-ICP-DRC-MS

method and (b) fractions from SAX separation were analysed

by CE-ICP-DRC-MS. Species identification was regarded as

OK when species matched the standard compounds after

serial analysis with both chromatography/electrophoretic

techniques (match in first and second technique).

Results and discussion

Quality control results

Checking total Se determination by analysis of urine and serum

control materials resulted in Se concentrations of 61±3 μ g/L for

serum or 24±3 μ g/L for urine. The manufacturer ’s target mean

values of 62 μ g/L for serum and 23 μ g/L for urine were found.

Mass balances during SAX-ICP-DRC-MS were calculat-

ed as described above. Recoveries were 106±11 % for

serum, 96±9 % for CSF, 106±10 % for GPx, 97±8 % for

Se IV, 102±8 % for TrxR, 89±8 % for Se-HSA, and 84±

13 % for SePP. The lower recovery for SePP is explained by

stability problems accompanied by an increasing Se VI

signal, both already observed previously [18].

Species identification using two 2D approaches

A) Figure 1 shows chromatograms of two SEC fractions

from serum (mass range 66 – 44 kDa, containing SePP,

Se-HSA and (traces of) TrxR or from CSF (mass range

95 – 80 kDa, containing GPx). These SEC fractions were

analysed by SAX-ICP-DRC-MS, i.e. serially first char-

acterized by SEC and second by SAX. In the 66−44 kDa

SEC fraction, SePP (3.5 min), Se-HSA (23.5 and

37.5 min, see below) and traces of TrxR (19.5 min) are

eluting at their specific SAX retention times. Thus, these

proteins appeared in the “correct ” size fraction and at

their specific retention times in SAX. Analogous, in the

95−80 kDa fraction from CSF, human GPx (86 kDa) was

collected and appeared subsequently at its typical

Table 1 The instrumental settings for ICP-DRC-MS

Instrument Perkin Elmer NexIon DRC

Plasma conditions

RF power (W) 1,250

Plasma gas flow (L/min) 15

Auxiliary gas flow (L/min) 1.05

Nebulizer gas flow (L/min) 0.98 daily optimized

Nebulizer (optimal flow rate

according to provider)

Meinhard low flow (300 μ L/min)

Mass spectrometer settings

Dwell time (ms) 300

Sweeps per reading 6

Readings per replicate 1,000

Autolens On

Ions montored 78Se, 80Se, 103Rh

Reaction gas CH4

Reaction gas flow rate (mL/min) 0.6

Rejection parameter, q 0.6

Rejection parameter, a 0


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retention time in SAX. This human GPx fraction also co-

eluted with a GPx standard, commercially prepared from

bovine erythrocytes. Therefore, the latter was used as RT

standard for GPx from our human samples.

B) For further strengthening, the peak identification SAX

fractions were analysed with CE-ICP-DRC-MS, too. As

an example, Fig. 2 shows electropherograms of two

fractions of regular SAX separation from serum, collect-

ed at typical RT of GPx or of TrxR and Se IV. Observed

peaks match the migration times of respective standard

compounds. By both approaches, we found a match in

both used techniques (SEC-SAX or SAX-CE). Species

identification therefore was regarded as sufficient.

Results on Se species in serum and CSF

According to our previous experience from [18], where

several peaks were eluting close to each other and

consequently identification with standard-matched method

was partly difficult and time-consuming, we changed the

elution gradient and flow rate to values given in the “Ex-

perimental” section. This resulted in a flatter gradient in the

mid-chromatogram and peaks appeared with more distance

to each other. With this optimized method, the subsequent

analysis of Se standard mixtures, serum and CSF samples

were performed. Figure 3 shows examples of chromato-grams from CSF and serum.

Notably, Se-HSA showed two elution peaks, the first

with a narrow peak shape at 25.4 min (where it had been

expected from previous experiments) and surprisingly a

second broader elution at 37.2 min. This observation was

made first with Se-HSA single standards but also in samples

(CSF and serum) with and without standard addition, i.e. the

two peak signals were seen in all sample types in both, UV-

and ICP-MS detection. Therefore, the sum of both peaks

was considered as “total Se-HSA”. A double peak for HSA

was shown also by Xu et al. (there Fig. 1) [19]. The method

of these authors was taken as a basis for our Se speciationmethod. Aside from identified compounds, such as SePP,

SeM, GPx, SeC, TrxR, Se IV, Se-HSA and Se VI also

unknown peaks (“U”0unknowns) were monitored in some

of the samples, where no standards matched the retention

times or met peaks after standard addition. Table 2 shows

the Se concentrations of total Se, the median and concentra-

tion range (minimum, maximum) of Se species from 24

paired serum and CSF samples.

For both, total Se from serum and CSF, the values were

lower than found in our previous studies from 2009 and

2011 [17,18], but are in agreement with other findings e.g.

from reference [14]. The presence of SePP in both sample

types was to be expected as SePP had been found in serum

and CSF previously. In this study, SePP concentration in

serum is less compared to findings of other authors [19,22].

In turn, Se IV in part showed considerable amounts, which

could point to stability problems of SePP, observed already

previously [18]. In CSF, however, the median of 0.474 μ g/L

Se from SePP corresponds roughly with 15 μ g/L SePP

(whole protein) from a former, preliminary approach

(Schweizer and co-workers (Ulrich Schweizer, J. Köhrle,

B. Michalke, unpublished results).

Scharpf et al. [14] found SePP-CSF independent from

SePP-serum, locally expressed in the brain. Further, SePP

seems to play an important role in neuronal survival by

protection against reactive oxygen species (ROS) [24]. The

presence of TrxR fits well to the supposed protective action

against ROS [25]. The primary defense line against oxida-

tive stress is based on superoxide dismutase activity, which

however, is resulting in H2O2 production. Subsequently,

peroxides are eliminated by the activity of the sele no-

enzymes TrxR and GPx, both being known to be expressed

in brain of animals [10]. Aside from TrxR, this time we also

0

1

2

3

4

5

6

SEC fraction from serum: MW 66 kDa - 44 kDa, containing:

SePP, Se-HSA (1+2), [traces of TrxR]

8

0 S e / 1 0 3 R h

8 0 S e / 1 0 3 R h

min

0.3

0.5

0.7

0.9

0 10 20 30 40 50

0 5 10 15 20 25 30 35 40 45

min

SEC fraction from CSF: MW 95 kDa - 80 kDa, containing: GPx

grey line: GPx standard

1

(2)

3

4

5

Fig. 1 Species identification by a 2D approach: mass range-

characterized SEC fractions from serum or CSF were subsequently

analysed by the SAX-ICP-DRC-MS method. Retention time matcheswere found in SEC (correct mass fraction) and in SAX (correct RT).

Peak assignment (top): 10SePP, (2)0traces of TrxR, 30Se-HSA-1, 40

Se-HSA-2. ( Bottom): 50GPx, compared to GPx standard


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found GPx. This was an improvement compared to our inves-

tigations in 2011, where we had observed stability problems

for GPx [18]. The presence of GPx is in accordance with Arnér

et al. [25], who had determined GPx in brain. Pyne-Geithman

et al. [26] analysed GPx in CSF, too. In serum, GPx was

already determined from several authors previously in a range

of ca. 10 – 18 μ g/L [2,19]. The GPx Se concentration found in

this study strives this range at the lower level (cf. Table 2).

The finding of Se-HSA in CSF was also not surprising.

HSA is not synthesized in the brain, but crosses NB to a certain

extent. Actually, the HSA quotient of CSF/serum is the stan-

dard mean to evaluate the intactness of the barrier. Se-HSA is

generally accepted for serum [e.g. 19,27,28]. Therefore Se-

HSA, like HSA, is transported in small amounts across NB

into CSF. Jitaru et al. [22] presented Se-speciation in serum

where Se-HSA amounted to 19 μ g/L. This is in accordance to

the median value from this study being 18.4 μ g/L.

To understand in which form selenium is crossing the NB,

Se species median values from serum and CSF were taken

from Table 2 and relationships were calculated. First, this was

done for Se-species-serum vs. Se-total-serum to elucidate which

of the Se-compounds mainly account for Se-total concentration

in serum. Relationships were evaluated according to their

linear regression coefficients, where r 2 was assigned to:

<0.10no relationship, independent; 0.1 – 0.250low influence;

0.25 – 0.40some influence; 0.4 – 0.50clear influence; 0.5 – 0.60

high influence; >0.60leading influence.

In serum, it became apparent that Se-total concentration vs. Se-

HSA (r 200.011), or vs. Se IV (r 200.0099), or vs. Se VI (r 20

0.0088) and vs. GPx (r 200.0265) did not show linear correla-

tion. The influence of those Se species on total Se concentration

in serum seemed to be low. Contrary, this was different for

SePP: a leading influence was seen for SePP-serum on total Se-

serum (r 200.9332), however, only as long as total Se was >65μ g/

L in serum. Below this concentration, no clear relationship could

be confirmed (r 200.0229). This is shown in Fig. 4.

The strong relationship between SePP-serum and total Se-

serum is known in general, since SePP is known as main Se

species in serum [19]).

When elucidating Se-species-serum vs. Se-species-CSF, the

independence of total Se-CSF from total Se-serum found pre-

viously (r 200.0001, [17]) was confirmed (this time, r 20

0

500

1000

1500

2000

2500

3000

3500

0 5 10 15 20

SAX-fraction (19-21 min) containing TrxR and Se IV

8 0 S e , c p s

8 0 S e

, c p s

min

SAX fraction

Se IV and TrxR standard

Se IV

TrxR

0

500

1000

1500

2000

2500

3000

0 5 10 15 20

SAX fraction (17 - 19 min) containing GPx

min

SAX fraction

GPx standard

Fig. 2 Species identification by

another 2D approach: Fractions

of SAX separation from serum

are analysed by CE-ICP-DRC-

MS. Top: The SAX fraction at

GPx-retention time shows also

matched peak in CE-ICP-DRC-

MS analysis. Bottom: The SAX

fraction at TrxR and Se IV-

retention time shows matched peaks in CE-ICP-DRC-MS

analysis. Retention time

matches thus are found for SAX

(correct RT/fraction) and CE

(correct migration time). For

improved visibility standard

electropherograms (grey) are

plotted with an off-set


7/25/2019 art%3A10.1007%2Fs00216-012-6294-y


0.0002). Similarly, SePP-CSF was independent of SePP-serum

(r 200.0365). This result fits well to the local SePP expres-

sion in brain being independent of SePP-serum, reported by

Scharpf et al. [14].

As for SePP, no linear relationship between serum and

CSF could be found for several of the remaining Se-

compounds, except however for GPx, Se-HSA and TrxR.

For these latter compounds, positive linear relationships

0

1

2

3

4

5

6

7

min

S e / R h

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20 30 40 50

0 10 20 30 40 50

min

S e / R

h

CSF

( S e M )

S e P P

S e C

U

S e C

G P x

S

e I V

S e - H S A 1

S e - H S A 2

S e V I

T r x R

S e I V

S e P P

U

T r x R

G P x

S e - H S A 1

S e - H S A 2

S e V I ( S

e M )

serum

Fig. 3 Examples of

chromatograms from serum and

CSF samples are seen. In total,

paired samples from 24

individuals (a ’ 2 replicates)

were analysed. Replicate

measurements resulted in equal

chromatograms

Table 2 Concentrations of Se species in serum and CSF

Serum total Se SeP SeM U1 U2 U3 U4 GPx SeC TRxR Se(IV) HSA Se(VI)

Se, μ g/L Min 36.22 1.55 <LoD <LoD <LoD <LoD <LoD <LoD <LoD <LoD <LoD 2.31 <LoD

Mean total Max 91.60 50.60 19.59 7.40 8.73 9.00 22.10 9.56 19.33 32.11 24.68 29.85 2.62

59.67 Median 58.39 5.19 0.23 <LoD <LoD <LoD 6.34 4.27 <LoD 1.64 12.25 18.03 <LoD

CSF, Se, μ g/L Min 0.274 0.040 <LoD <LoD <LoD <LoD <LoD <LoD <LoD <LoD <LoD <LoD <LoD

Mean total Max 2.371 1.213 0.116 0.352 0.562 <LoD <LoD 0.334 0.035 0.296 0.534 0.647 0.750

0.958 Median 0.861 0.474 <LoD <LoD <LoD <LoD <LoD 0.036 <LoD 0.035 0.046 0.068 <LoD

Ranges are given as minimum and maximum concentrations; additionally, the median values are shown. LoD, calculated as 3×SD of noise, was in

the range of 0.026 – 0.031 μ g/L for all investigated species and thus was set uniformly to 0.032 μ g/L


7/25/2019 art%3A10.1007%2Fs00216-012-6294-y


were observed. Still only small r 2 values were calculated for

Se-HSA-serum vs. Se-total-CSF (r 20

0.1650) or vs. Se-HSA-CSF

(r 200.1722). This can be explained by the regular NB func-

tion based on barrier integrity, which is usually expressed byQ-Alb, which is the quotient of HSA -CSF/HSA-serum. Q-Alb

values from our samples (median, 5.25×10−3) were in the

normal range for healthy adults (age-dependent range, 3×

10−3 to 8×10−3, [29]) and thus, only very little amount of

Se-HSA (or HSA in general) could pass NB. Protein transfer

from blood to CSF is restricted by the laws of diffusion and

consequently influenced by molecular size [30].

Fig. 4 The relationship of

SePP from serum to total Se

concentration in serum is

shown: A leading influence is

seen for SePP-serum on total Se-

serum (r 200.9332), as long as

total Se is >65 μ g/L. Below this

concentration, no clear rela-

tionship could be confirmed

(r 20

0.0229)

GPx grouped means: CSF vs. serum

y = 0.0091x + 0.0044

R2 = 0.7348

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

GPx in serum, µg Se/L

G P x i n C S F , µ g S e / L

y = 0.0188x - 0.0013

R2 = 0.8193

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.00 2.00 4.00 6.00 8.00 10.00 12.00

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

TrxR grouped means: CSF vs. serum

TrxR in serum, µg Se/L

T r x R i n C S F , µ g S e / L

Fig. 5 x – y plots are shown for GPx and TrxR, where grouped-mean

values (0 – 0.5, 0.5 – 1, 1 – 1.5…μ g/L) from serum-borne Se species are

related to the means of the respective CSF-borne Se species. This

allowed showing the relationships of GPx or TrxR between serum

and CSF more clearly. r 2 values are 0.7348 (GPx) and 0.8193

(TrxR). Slopes are 0.0091 (GPx) or 0.0188 (TrxR), which demonstrate

a moderate influence of GPx-serum on GPx-CSF, but a more profound

influence of TrxR -serum on TrxR -CSF

Q-Se-species

0

0,005

0,01

0,015

0,02

0,025

Q-GPx Q-TrxR Q-Se (IV) Q-Se-

HSA

Q-Alb

m e a s u

r e f o r

N B i n

t e g r i t y

8.31 x 10-3

21.34 x 10-3

3.82 x 10-3 3.85 x 10-3

5.25 x 10-3

Q [ m e d i a n ( C S F ) / m e d i a n ( s e r u m ) ]

Fig. 6 The Q values (ratios of Se species: CSF/serum) of median

values from Se species are shown for those Se compounds which had

median values >LoD. These values are compared to Q-Alb, which is the

clinical evaluation parameter for NB integrity. Further information

about the passage of Se species across NB is gained. Since SePP is

accepted to be expressed in brain independently from serum, it is not

considered here


7/25/2019 art%3A10.1007%2Fs00216-012-6294-y


In contrast to Se carriers, such as SePP or Se-HSA, the

linear relationships of Se enzymes protecting against oxida-

tive stress were stronger expressed: a clear linear relation-

ship could be confirmed for GPx-serum vs. GPx-CSF

(r 200.3837). GPx together with TrxR -serum are known as

typical Se enzymes protecting against oxidative stress

[15,16,25,26]. Interestingly, TrxR also showed a consider-

able linear relationship between serum and CSF: TrxR revealed a correlation coefficient at a comparable level as

GPx when related to Se-total-CSF (r 200.3836), but r 2 was

significantly higher, reaching a value of r 200.6293, when

being related to TrxR -CSF. This confirmed a leading influ-

ence of TrxR -serum on TrxR -CSF.

However, the r 2 values determined for GPx and TrxR

appeared lower than expected from the x – y plots which

showed apparently clear relationships. This was suspected

to be due to high variance of those values which were close

to LoD. For compensating variances of values close to LoD

but maintaining slope and equation, an additional evaluation

model was used, where grouped-mean values (0 – 0.5, 0.5 – 1,1 – 1.5…μ g/L) from serum-borne Se species were related to

the means of the respective CSF-borne Se species. This

resulted in x – y plots showing the same relationships clearer

with r 2 values of even 0.7348 (GPx) and 0.8193 (TrxR).

Slopes were the same as without grouped-mean calculations

and demonstrated an influence of GPx-serum on GPx-CSF, and

again a leading influence of TrxR -serum on TrxR -CSF. Figure 5

shows the y-x-plots of grouped-mean values relationships

for GPx and TrxR.

The concentration ratios (CSF/serum) were calculated sim-

ilar to Q-Alb for those Se species which were regularly detect-

able in both sample types, i.e. where median values were >LoD,

to get further information about their passage across NB. Since

SePP is accepted to be expressed in brain independently from

serum, this Se species was not considered here.

The ratios are shown in Fig. 6 and compared to Q-Alb.

With an intact neural barrier (Q-Alb in normal range, as in

our paired samples) protein transfer from blood to CSF is

restricted by the laws of diffusion and consequently influ-

enced by molecular size [30]. Therefore, HSA, having a

molecular size of 66 kDa, is crossing NB only in small extent

with a Q-Alb of 5.25×10−3. However, the increased ratios of

TrxR (55 kDa) and GPx (86 kDa) are not explained by a

molecular size dependence, as GPx is even larger than Se-

HSA and TrxR ratio is increased much more than expected

regarding the somewhat smaller molecular mass. This is clear-

ly seen when calculating molecular mass dependence at NB

from Q values of various proteins and applying this value to a

molecular mass of 55 kDa (TrxR): From molecular masses (of

proteins) and Q values provided by Reiber et al. [29] a simple

calibration of Q vs. ln(molecular mass) is calculated, resulting

in the linear equation: y0−0.2649 x+5.5736. Based on this

equation the Q value for 55 kDa (TrxR) is 5.91×10−3, which

is close to Q-Alb, but significantly lower than the actually

measured Q-TrxR of 21.34×10−3.

Thus, we hypothesize, that there could be a facilitated

diffusion across NB for the anti-oxidative Se-enzymes GPx

and TrxR or that they might be additionally expressed in brain.

Conclusion

For the investigation of selenium species distribution on

both sides of NB, 24 individual paired serum and cerebro-

spinal fluid samples from 24 neurologically healthy persons

were analysed. SAX-ICP-DRC-MS was optimized and used

for analysis. Species identification was proofed by use of

two 2D approaches. Linear regression coefficients were

calculated to evaluate relationships between species concen-

trations present in serum and CSF, as well as between

species concentration and total selenium content.

In serum, a leading influence on total Se -serum was seen

only for SePP-serum when total Se was >65 μ g/L. The

previously described independence of total Se-CSF on totalSe-serum and of SePP-CSF on SePP-serum was confirmed.

Linear relationships of Se compounds between serum

and CSF could be found only for GPx, Se-HSA and TrxR

with varying r 2 values. Q-Alb values from our samples were

in the normal range for adults. However, the increased Q

values of TrxR and GPx are not completely explained by a

molecular size dependence, as GPx is even larger than Se-

HSA and TrxR ratio is increased much more than expected

regarding the somewhat smaller molecular mass. Thus, we

suggest that there may be a facilitated diffusion across NB

for GPx and TrxR or that they might be additionally

expressed in brain.

Acknowledgments The authors thank Katharina Fernsebner for

reading the manuscript.

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