Enriched stable isotopes and isotope pattern deconvolution for quantitative speciation of endogenous...

PAPER www.rsc.org/jaas | Journal of Analytical Atomic Spectrometry

Enriched stable isotopes and isotope pattern deconvolutionfor quantitative speciation of endogenous and exogenous seleniumin rat urine by HPLC-ICP-MS†

H. Gonz�alez Iglesias,a M. L. Fern�andez S�anchez,a J. A. Rodrı́guez-Castrill�on,a J. I. Garcı́a-Alonso,a

J. L�opez Sastreb and A. Sanz-Medel*a

Received 12th November 2008, Accepted 2nd February 2009

First published as an Advance Article on the web 2nd March 2009

DOI: 10.1039/b820297b

Interest in selenium metabolism and its role in health and disease continue to stimulate a lot of

bioanalytical research. The use of enriched stable isotopes of Se and isotope pattern deconvolution

(IPD) with an ICP-MS in lactating rats is approached here for total selenium distribution studies. A

mathematical tool has been developed to calculate supplemented and endogenous total Se contents in

urine and faeces by ICP-MS. Also, quantification of endogenous (natural) and exogenous seleno-

species by HPLC-ICP-MS has been worked out. The proposed IPD methodology, for total

determinations and for quantitative speciation, was applied to reference materials to validate total

selenium quantification in faeces and quantitative selenium speciation in urine samples. Selenium

apparent absorption and retention and natural and exogenous selenium distribution in urine samples

were calculated. Interesting quantitative information about selenium bio-transformations and final

catabolism can be obtained from careful examination of the results observed, using such analytical

tools, of the urinary endogenous and exogenous selenium metabolites in urine of rats and their kinetics

throughout the two weeks of selenium supplementation.

Introduction

Selenium (Se) is an essential trace element that occurs in seleno-

proteins in the form of selenocystine. Most of the currently

known selenoproteins are selenium-dependent enzymes,

including glutathione peroxidases, deiodinases and thioredoxin

reductase, with antioxidant functions. Interestingly, even

without being incorporated into proteins, selenium can modulate

the activities of certain transcription factors and kinases.

Through its association with enzymes, Se is involved in the

catabolism of peroxides generated during lipid oxidation. Other

selenoproteins help to regulate thyroid function and play an

important role in the immune system.1 Moreover, at concentra-

tions higher than nutritional requirements, selenium has anti-

cancer effects and so is considered a typical ‘‘nutraceutical’’

element.2

Interest in the metabolism of Se and its function in health and

disease, continues to stimulate much research these days.3

Radioactive isotopes began to be very popular on the 1940s to

study mineral absorption and metabolism in humans and

animals, but this approach has decreased in popularity in recent

years because of radioactivity risk.4,5 By contrast, the use of

enriched stable isotopes as metabolic tracers with MS detectors

has extended rapidly in the past two decades, particularly in

aDepartment of Physical and Analytical Chemistry, Faculty of Chemistry,University of Oviedo, Julian Claverı́a 8, 33006 Oviedo, Spain. E-mail:[email protected]; Fax: +34 985103125; Tel: +34 985 103474bDepartment of Neonatology, Hospital Central de Asturias, Oviedo, Spain

† This article is part of a themed issue dedicated to Professor Jean-MichelMermet, in recognition of his contributions to the field of atomicspectrometry.

460 | J. Anal. At. Spectrom., 2009, 24, 460–468

human nutrition studies.6,7 Enriched stable isotopes are crucial to

study the fate of minerals and trace elements in biological

systems. Isotope tracer experiments are very similar to radio-

isotope trace experiments, except that enriched stable isotopes

tracers have a number of advantages over radioisotopes,

including: (i) no exposure to radiation occurs, consequently they

can be used in high-risk population groups such as infants,

children, and pregnant or lactating women;4,6 (ii) some minerals

have no radioisotope that can be used satisfactorily as a tracer;

(iii) the possibility exists of using multiple stable isotopes of

a given element; (iv) multiple isotopes of multiple different

elements can be administered simultaneously or sequentially to

investigate synergist or detoxification effects of a given element

on the biological functions of others and (v) ICP-MS wide

implementation today allows for a sensitive detection and

determination of such stable isotopes.

In this sense, selenium stable isotopes in connection with ICP-

MS have been used extensively to study selenium metabolism in

rats and in humans and its bioavailability from food in faeces or

in urine.8 Dual isotope ratio measurements require selenium

equilibration between natural existing selenium in the sample

and added isotopically enriched Se.8 This is particularly so prior

to the administration of any other isotopically enriched tracers,

to simplify the calculation procedures. In this work we have

applied isotope pattern deconvolution (IPD) for isolating

distinct isotope signatures from mixtures of natural abundance

and enriched tracers.9,10 This technique has proved to be an

important tool in nutritional studies and has recently been

applied in depth to total elemental analysis11–13 and to speciation

analysis as well.10,14 By measuring isotope ratios by ICP-MS and

using IPD quantitative data of endogenous and exogenous

This journal is ª The Royal Society of Chemistry 2009

essential (or toxic) elements and information on their metabo-

lism, without resorting to prior isotope enrichment procedures,

can be obtained.12,15

In this work we explore further the application of isotope

pattern deconvolution (IPD) to discriminate the fate of endog-

enous and supplemented selenium and their catabolism species in

rat urine, by using a collision cell ICP-MS12 coupled to an HPLC

separation. Such set-up in combination of two different enriched

isotopes: a ‘‘metabolic’’ tracer (77Se) and a post-column ‘‘quan-

titation’’ tracer (74Se), allows the quantitative discrimination of

‘‘natural’’ and ‘‘enriched’’ selenium species in rate urine by

resorting to IPD calculations.12

Experimental

Instrumentation

An ICP-MS model Agilent 7500ce (Agilent Technologies,

Tokyo, Japan) equipped with an octapole reaction cell was used.

The operating conditions are given in Table 1 for the time

resolved and isotope dilution analysis modes.

For identification of selenospecies an electrospray Q-TOF

mass spectrometer model Qstar XL (Applied Biosystems,

Darmstadt, Germany) equipped with the ion spray source using

N2 as nebuliser gas was used. The instrument was calibrated

every day using a standard solution of poly(propylene glycol).

Samples are injected using a syringe pump at a flow rate of 5 mL

min�1. The scanned range was from 100 to 500 m/z and the

applied voltage 5500 V.

The High Performance Liquid Chromatography system used

consisted of an HPLC pump (Shimadzu, model LC-20AD

(Kyoto, Japan), a Rheodyne six-port injector (CA, USA) with

a 100 mL sample loop and a column.

Reversed-phase (RP) LC was performed on a Mediterranea

Sea18 column (150 � 4.6 mm, 5 mm) (Teknokroma, Barcelona,

Table 1 ICP-MS operating conditions and data acquisition parameters

Plasma parametersRF power/W 1500Plasma gas flow rate/l min�1 15Auxiliary gas flow rate/l min�1 1.1Sampling depth/mm 5.8Ion lens setting Daily optimised for best sensitivity

of 10 mg l�1 Li, Co, Y and TlReaction cell parametersH2 gas/ml min�1 4aOctapole bias/V �13aQP bias/V �12Data acquisition parameters (IPD analysis)Acquisition mode ID analysisMonitored isotopes 74, 76, 77,78, 79, 80, 81, 82, 83Points per peak 3Acquisition time per point/s 4Replicates 5Data acquisition parameters (IPD-Post column)Acquisition mode Time resolved analysisMonitored isotopes 74, 76, 77,78, 79, 80, 81, 82Pints per peak 1Integration time (per peak)/s 0.3

a The ion lens voltage was ‘‘tuned’’ daily with a 100 ng g�1 seleniumsolution to obtain a maximum signal for selenium.


Spain). For anion exchange experiments a Mono Q 5/50 GL

(Amersham Biosciences, UK) column was used.

Sample digestions were carried out with a Milestone micro-

wave oven model Ethos-1 (Microwave Laboratory Systems,

Socisole, Italy).

Sample preconcentration was performed on a Speed Vac

Concentrator 5301 (Eppendorf, Hamburg, Germany).

Reagents and materials

Enriched 77Se (94.75 abundance of 77Se) and 74Se (98.84 abun-

dance of 74Se) were obtained from Cambridge Isotope Labora-

tories as elemental Se powder and dissolved in a minimum

volume of sub-boiled nitric acid and then diluted with ultra pure

water, as required. Enriched 76Se (74.89 abundance of 76Se) was

obtained from LGC Limited (Teddington Middx, UK) as Sele-

nomethionine standard and dissolved 0.5 mg of the solid in

15 mL of ultrapure water. Selenium concentration in these

solutions was determined by reverse isotope dilution analysis

using a natural abundance (49.61 abundance of 80Se) Merck

certified Se standard (Merck, Darmstadt, Germany). Biological

reference materials analysed in this work were: Seronorm�Trace Elements Urine LOT N�2525 (SERO AS, Billingstad,

Norway) and NIST SRM 1577a Bovine Liver.A commercial

formula milk containing low amount of Selenium (50 � 5 ng g�1

powder) was used for rat feeding.

Distiled de-ionized water (18 MU cm) was obtained by means

of a Mili-Q system (Millipore).

Formic acid, methanol, acetonitrile and ammonium acetate of

analytical grade were also used.

Animals

The animal experiments was carried out in the Animal Unit

Laboratory of the University of Oviedo, following the guidelines

established by The Local Ethics Committee (R. D. 223/1988) and

The Animal Experiments Directive (86/609/EEC) on the

protection of animals used for experimental and other scientific

purposes. Two-week-old wistar rats were maintained in meta-

bolic cages (three rats/cage) at 22 �C with a light/dark cycle of 12/

12 h for two weeks. Afterwards, a group of three of those rats,

weighting 30.0–31.0 g, was fed with reconstituted formula milk

ad libitum for another two weeks. Another group of three rats,

weighting 29.6–30.6 g, was fed with the same reconstituted

formula milk but supplemented with 77Se at a concentration of

0.15 mg Se/g powder milk (level recommended for human nutri-

tion16) ad libitum, for two weeks. For one day, 77Se intake was

calculated by difference between total milk (0.15 mg 77Se/g

powder milk) for the three rats and the remaining milk weight

after 24 h (that is the non-ingested milk). Urine was collected

from the metabolic cage every 24h, and faeces were recovered as

they appeared in the special receptacle under the metabolic cage.

The final weight of the rats after the feeding period ranged

between 77.44 and 85.30 g for the supplemented rats, and

between 70.30 and 74.60 g for the non-supplemented.

At the end of the supplementation period blood samples

were obtained from all the rats under light isofluorane anaes-

thesia, and the clotted blood was centrifuged at 1600g for

10 min to obtain serum samples. Faeces sample were

J. Anal. At. Spectrom., 2009, 24, 460–468 | 461

lyophilised. The serum, urine and faeces samples were stored at

�80 �C until use.

Procedures

Sample digestion. The total concentration of selenium and the

fraction of total endogenous and exogenous selenium in faeces

(and the bovine liver reference material used for just validation

purposes) were determined by ICP-MS after microwave diges-

tion. Approximately 0.1 g of faeces powder (or bovine liver

reference material) was placed into a PTFE digestion vessel and 9

mL of sub-boiling nitric acid (30%), 1.5 mL of 30% suprapur

hydrogen peroxide, and appropriate amount of 74Se enriched

spike were added. The vessels were immediately closed after the

addition of the reagents. A microwave program previously

developed in our group for biological material was applied.17 At

the end of the digestion, the resulting solutions were made up to

20 mL with ultrapure water to be finally analysed by ICP-MS.

The optimum 74Se spike amounts for quantification were

evaluated by using the random error propagation theory. Taking

into account the natural abundance of Se and the isotope

composition of the 74Se spike, the optimal isotope ratio 78Se/74Se

turned out to be 0.36. According to the level of Se expected in the

sample, the amount of 74Se spiked is calculated to obtain an

optimum ratio 78Se/74Se (range 0.1 to 1, optimum value 0.36).17

Isotope pattern deconvolution. Consider a sample containing Se

of natural abundance to which two isotopically enriched spikes

(74Se as quantitation tracer and 77Se as metabolic tracer) has been

added. The isotopic composition measured in this blend for the

Se isotopes 74, 76, 77, 78, 80 and 82 could be decomposed into

three components, corresponding to: a) the isotopic composition

of natural selenium (Senat), b) the isotopic composition of the

isotopically enriched 77Se (Seenr77) spike and c) idem for 74Se spike

(Seenr74), by using the following matrix equation:

266666666664

74Ab

76Ab

77Ab

78Ab

80Ab

82Ab

377777777775

¼

266666666664

74Senat74Seenr77

74Seenr74

76Senat76Seenr77

76Seenr74

77Senat77Seenr77

77Seenr74

78Senat78Seenr77

78Seenr74

80Senat80Seenr77

80Seenr74

82Senat82Seenr77

82Seenr74

377777777775

�

2664

xSenat

xSeenr77

xSeenr74

3775þ

266666666664

74e

76e

77e

78e

80e

82e

377777777775

The values of xSenat, xSeenr77

and xSeenr74correspond to the molar

fractions of selenium and are found by least square minimisation

of the error vector ‘‘e’’. Once the molar fractions of the element

and their uncertainties are calculated by the multiple linear

regression, the total amounts of natural abundance and 77-

enriched selenium can be easily calculated since the amount of74Se added is known.12

The same procedure can be applied for quantitative speciation

of endogenous and exogenous selenium by using post-column

isotope dilution analysis by using 74Se as ‘‘quantitation’’ tracer.

As the mass flow of 74Se is known, the mass flows for natural

abundance selenium species and 77Se can be calculated by

applying the IPD procedure to every point of the chromatogram.


The correction for mass bias and spectral interferences described

before11,12 will need to be applied to every point in the chro-

matogram.

Determination of natural and endogenous total Se in faeces by

IPD-ICP-MS. Digested faeces containing natural and exogenous

Selenium and the appropriate amount of an enriched 74Se stan-

dard solution were analysed by ICP-MS. Six selenium isotopes

(74, 76, 77, 78, 80 and 82) in the sample were monitored by ICP-

MS under the conditions specified in Table 1. Then, after

adequate corrections of dead time, internal correction of SeH+ or

BrH+ interferences, and of the mass bias,11 IPD methodology

previously developed for Se12 was used to calculate the concen-

tration of ‘‘endogenous’’ (Senat) and ‘‘supplemented’’ (enriched77Se) selenium present in the faeces.

Natural and endogenous Se speciation in urine by HPLC-IPD-

ICP-MS. Chromatographic separation of Se species in urine

samples was carried out in a C18 HPLC column coupled on-line

to the ICP-MS (operating conditions are given in Table 1). Urine

samples were filtered and diluted (1 + 1) previous to their injec-

tion in the HPLC system. The sample injection volume was 100

mL and the mobile phase was 100 mM NH4Ac with pH 4.5 in 5%

of methanol at a flow rate of 1 ml/min.18

The quantification of Se species eluting from the column was

performed by post-column IDA. A 74Se-enriched standard

solution of the appropriate concentration was continuously

introduced (at 0.1 ml min�1) using a peristaltic pump at the end of

the column through a T piece. The emerging equilibrated mixture

of isotopes was nebulised into the plasma. The intensities in the

chromatogram (counts s�1) were converted into two independent

natural and exogenous (supplemented) Se mass flow chromato-

grams (ng min�1), by applying the IPD procedure described

before.12 Finally, the amount of each selenospecies, containing

natural or exogenous selenium, was determined in each chro-

matographic peak by area integration and IPD.

Identification of selenospecies in urine. Lyophilised urine (150

mL) was extracted in 15 mL of methanol in an ultrasonic bath for

10 min. The extract was centrifuged 5 min at 5000 g and the

supernatant was evaporated to dryness in a stream of nitrogen at

ambient temperature and reconstituted in 1 mL of eluent for RP

column.18 A volume of 100 mL was injected in the RP-C18

column. The elution was carried out with 100 mM ammonium

acetate, 5% MeOH, pH ¼ 4.5 at flow rate of 1mL/min.

The eluting fractions, at retention time of 7.8 min for 10

consecutive injections, were collected desalted and lyophilised.

The lyophilized was dissolved in 100mL of 50% ACN, 0.2%

formic acid. The solution was introduced into the electrospray at

a flow rate of 5 mL min�1. The instrument was operating in the

positive ion mode. The applied voltage to the capillary was 5500

V, and the collision energy was around 35 V. N2 was used as the

nebulizing gas. The ESI Q-TOF mass spectra of selenium-con-

taining peptides were acquired in the range m/z 100–500.

For the identification of inorganic selenium, anion exchange

chromatography was also used as a complementary separation.

The fraction eluting in C18 column at retention time of 2.7 min

(five successive injections) was collected and preconcentrated in

a speed vac and a 100 mL volume of the preconcentrated solution


was injected in the Mono Q column. The mobile phase and the

gradient used for elution were: A phase (10 mM NH4Ac, pH ¼7.0); B phase (500 mM NH4Ac, pH ¼ 7.0); gradient: 0 to 10 min;

0 to 100% B phase. The flow rate used was 1 mL/min. The

identification of the major selenium species present in this frac-

tion was performed by matching retention time with that of the

corresponding inorganic selenium standard.

Results and discussion

Validation of the dual spike IPD procedure

A biological certified reference material (bovine liver, NIST

SRM 1577a) was analysed in order to validate the sample

digestion procedure and the isotope pattern deconvolution

approach developed for quantification of total natural and

exogenous selenium.12 The sample, which contains a certified

amount of Se with natural isotopic abundance, was spiked with

the two selected enriched Se isotopes (74Se and 77Se), before

microwave digestion. The amount of 77Se solution added was

considered as unknown and only the 74Se spike solution was used

for quantification. The selenium isotopes were monitorized by

ICP-MS in the digested samples. The concentration of natural

selenium (reference value) and exogenous Se (spiked 77Se) in the

reference sample can be determined from the selenium isotope

composition measured in the sample, the three isotope patterns

(the natural, the 77Se tracer and the 74Se spike) and applying the

IPD methodology.12 Final quantitative results obtained for the

bovine liver reference material are given in Table 2. As can be

seen, values for both natural Se (certified value) and for 77Se

obtained agreed very well with the Se present in the sample

(natural Se) and the 77Se tracer added.

Fig. 1 Cumulative faecal selenium (totalselenium for non supplemented

group and natSe and 77Se for the Se supplemented group) excretion during

the lactating period.

Application of a dual spike IPD to determine natural and

exogenous total Se in faeces and urine

The application of the dual spike IPD approach (described

before) was tested to study total natural and supplemented

selenium in rat faeces. The determination of both, natural and

Table 2 Analysis of Certified Reference Material for the validation of the IP

Reference Material

Natural selenium

Certified

NIST SRM 1577a Bovine Liver 0.71 � 0.07 mg/g

Table 3 Natural Se and 77Se intake and excretion (in urine and faeces) by th

Period (days)

Selenium intake (mg) Selenium excrete

natSe 77Se natSe

1–3 1.44 5.65 2.08 � 0.024–7 3.75 12.86 4.30 � 0.028–10 4.45 13.89 5.04 � 0.0111–14 5.19 12.40 6.56 � 0.03Total 14.83 44.80 17.98 � 0.02

a Average of total Se excreted in the summed-up urines, and faeces, of the th


supplemented selenium, in the real samples were carried out as

follows: 5 faeces samples of the non-supplemented rats and 8

faeces samples of the 77Se supplemented rats (see Procedures)

were spiked with a standard solution of 74Se before their ICP-MS

Se isotope abundances determinations. From those abundances,

isotope pattern deconvolution was applied to calculate the

amount of endogenous (natSe) and supplemented (77Se) selenium

present in the analysed samples. The data were then referred to

the total sample selenium (as determined by ICP-MS) to calcu-

late cumulative absorption and excretion data.

The obtained data for natural and exogenous selenium

excreted in urine and in faeces all along the lactating period

(grouped on three days stages) are given in Table 3. For faeces,

the cumulative results are plotted in Fig. 1 with reference to the

days during the study. Every data point corresponds to the mean

amount of excreted selenium (in nanograms) by the rats in each

group (three rats per group). As can be observed, a linear

D procedures (n ¼ 3)

77Se-enriched

Found Added Found

0.69 � 0.04 mg/g 0.65 mg/g 0.63 � 0.04 mg/g

e three supplemented rats during the 14 days studya

d in urine (mg) Selenium excreted in faeces (mg)

77Se natSe 77Se

0.90 � 0.01 0.024 � 0.005 0.001 � 0.0012.74 � 0.02 0.543 � 0.004 0.219 � 0.0023.79 � 0.02 0.087 � 0.004 0.054 � 0.0047.12 � 0.02 0.295 � 0.004 0.247 � 0.003

14.55 � 0.02 0.949 � 0.004 0.521 � 0.002

ree assayed rats (all living in the same metabolic cage, simultaneously).


Table 4 Supplemented selenium apparent absorption and retentionduring the study

Period (days)

Apparent Absorption Apparent Retention

mg 77Se % 77Se mg 77Se % 77Se

1–3 5.65 � 0.01 99.9 � 0.2 4.74 � 0.02 83.9 � 0.34–7 12.64 � 0.02 98.3 � 0.2 9.90 � 0.02 77.0 � 0.28–10 13.84 � 0.04 99.6 � 0.3 10.05 � 0.01 723 � 0.111–14 12.15 � 0.03 98.0 � 0.2 5.03 � 0.02 40.6 � 0.1Total 44.28 � 0.03 98.4 � 0.2 29.72 � 0.02 66.3 � 0.2

increase of excreted selenium in the non supplemented group (r ¼0.997, and a slope around 20) is observed. After fourteen days the

total natural selenium loss was about 0.22 mg. For the supple-

mented group the cumulative natSe and 77Se excreted during the

first 5 days were similar and close to values for the non-supple-

mented group (and exogenous Se was not detected). However,

the cumulative faecal excretion of 77Se and natSe here increased

abruptly after 6 days of starting the supplementation and then it

continued to increase linearly (see Fig. 1) again. It is worth

mentioning that less than around 1.2% of the ingested 77Se was

eliminated by faeces.

Endogenous faecal Se excretion (natSe) turned out to be quite

larger than that of exogenous excretion (77Se). Moreover, it was

much higher in the supplemented group than in the non-sup-

plemented one. This fact clearly indicates that a substantial

fraction of endogenous selenium is lost in the gastrointestinal

tract passage. Clearly, the cumulative loss of natural selenium

observed in the supplemented group was much higher than in the

non-supplemented group. In the 14th day the measured ratio Se77/

Senat for the supplemented group was 0.78 � 0.03.

Urine determinations of total natSe and 77Se by the proposed

procedure were described in a previous work of our laboratory.12

Table 3 shows the values obtained now for natSe and 77Se excreted

in urine in the supplemented group all along the 14 days exper-

iment. As before, each point corresponds to the observed mean

amount of selenium (natural and enriched) excreted by three rats.

It can be seen that Se-supplemented group showed natural and

exogenous selenium excretion in urine increasing with time,

being the amount of exogenous selenium lower than that for natSe

until the 11 day. Then, the amount of excreted enriched 77Se

became even higher than that for natural selenium. After the 14th

day, about 44.3% of exogenous and 55.6% of endogenous sele-

nium was excreted in urine (in this way, 95% of total selenium

excreted was through urine and only 5% through faeces). About

38.6 % of total 77Se ingested during the supplementation is thus

excreted through urine. Interestingly, at the end of the supple-

mentation period the ratio of Se77/Senat in urine approaches

those ratios measured in serum (Se77/Senat ¼ 1.33 � 0.04) for the

three rats, a higher value than that found in faeces (0.78 � 0.03).

Cumulative natSe excreted by the un-supplemented group

increased only slightly (Fig. 1), so observed endogenous quan-

titative excretion (about 200 ng of Se) was about 5 times lower

than that observed in the supplemented group (about 950 ng).

Fig. 2 Typical chromatogram obtained for selenium isotopes (74Se, 76Se,77Se, 78Se), in a sample for the supplemented group of rats.

Apparent selenium absorption and retention

Apparent absorption and retention of 77Se were defined as:

[intake - faecal excretion/intake x100] and [intake-(faecal excre-

tion + urine excretion)/intake �100].19 The values of such

parameters have been calculated as well and they are shown in

Table 4. As can be seen, apparent absorption was not signifi-

cantly different all along this 14 days study, with an average

value of 98.8 � 0.5%. However, retention percentage was about

84% the first days of supplementation lent then it decreased

gradually dropping sharply (from 67% to about 26%) after the

11th day.

Considering selenium metabolism basic concepts,20 labelled Se

should be absorbed and transported in blood to the liver while

only a small fraction seems to be excreted via urine. After 11


days, the amount of 77Se (supplement) and natural Se in urine

become similar. This fact could be related with the slow turnover

(metabolism and final catabolism) of selenoproteins from tissues

as brain, muscle and bones.20

Speciation and identification of selenocompunds in urine

The separation of selenospecies present in urine samples under

study was performed by RP high performance liquid chroma-

tography (HPLC) coupled to ICP-MS. A typical chromatogram

(see Fig. 2) illustrates the observed distribution of selenospecies

in a supplemented rat urine sample. As can be seen in the profile,

two major peaks were detected: one at a retention time of 2.7

minutes (which could correspond to inorganic selenium) and the

second one 7.8 minutes, which could be a selenosugar, probably

Se-methyl-N-acetylselenohexosamine that if the main urinary

selenium metabolite.3,21,22 Thus, the nature of this seleno-

compound eluting at 7.8 minutes (see Fig. 2) was investigated

further by ESI-Q-TOF in the pooled supplemented rat urine.

This fraction at 7.8 min was purified and preconcentred

following the procedure described previously and the obtained

ESI-MS spectrum for such urine main Se metabolite is shown in

Fig. 3. As can be seen, the MS signals showing a characteristic

isotopic pattern of selenium were observed at masses 296.7, 297.7

and 299.7 (corresponding to the enriched 77Se, 78Se and 80Se

respectively). The most intense peak occurred at mass 296.7 (that

is, corresponding to 77Se-containing peak, the most abundant


Fig. 3 ESI-QTOF of supposed selenosugar Se-methyl-N-acetylselenohexosamine enriched in 77Se (fraction eluting at 7,8 min, Fig. 6).

isotope in the Se supplement used) confirming the presence of

Se-methyl-N-acetylselenohexosamine.21

On the other hand, the use of chromatographic methods based

on different (complementary) interaction mechanisms may be

also helpful for species identification.23 Therefore, further

anionic exchange chromatography was used in order to investi-

gate the identity of possible inorganic selenium appeared in the

dead volume of the C18 column (Rt ¼ 2.7 min). Thus, the fraction

eluting at 2.7 min on the C18 column was collected, preconcen-

trated and then injected (see procedures) in a Mono Q column. A

major peak at 6.2 min was the main selenocompound detected,

and it matched the retention time of Se (IV). At the dead volume

of the Mono Q column, a minor selenocompound was also

detected (signal of around 5% of the total existing selenium).

Fig. 4 a: natSe and 77SeO32� distribution in human urine reference. b: natSe

and 76Se-methionine distribution in human urine reference.

The IPD-HPLC-ICP-MS method for quantitative speciation of

selenospecies

In order to validate our IPD-HPLC-ICP-MS methodology for

quantitative selenium speciation in urine, the urine reference

material (Seronorm� Trace Elements Urine) was used. The

reference material was spiked with enriched 77SeO32� or 76Se-

methionine and diluted 1:1 with ultra pure water. After the

on-line separation via column RP-C18, the quantification of

selenium in the different chromatographic fractions was per-

formed by post-column isotope dilution analysis using a solution

of enriched 74Se for quantification of the ‘‘natural’’ and ‘‘enriched’’

supplemented Se components previously separated. Finally, the

IPD technique12 was applied to each point of the chromatogram

and so the ‘‘mass flow chromatograms’’ for the natSe and 77Selenite

(or 76Se-Met) were obtained (see Fig. 4). The amount of selenium

(natural selenium and 77Selenite/or 76Se-methionine) in each frac-

tion was calculated by integration of the chromatographic peaks

using the OriginPro 7.5 SR0 software (Microcal Software Inc.,

Northampton, MA, USA). The obtained results are shown in

Table 5.


As can be seen, the results for both Se natural (certified value)

and 77Se (or 76Se-methionine) obtained agreed very well with the

Se present in the sample (natural Se) and the 77Se (or 76Se-Met)

tracer added. The final concentration of Selenium species in the


Table 5 Integration values for the natural and 77Se or 76Se (expected and found)

Sample spiked with enriched 77SeO32� Sample spiked with enriched 76Se-methionine

Natural Se (mg/L) Supplemented 77SeO32� (mg/L) Natural Se (mg/L)

Supplemented 76Se-methionine (mg/L)

Certified 71 � 7 Found 73 � 4 Added 25 Found 26 � 2 Certified 71 � 7 Found 72 � 2 Added 19 Found 20 � 2

reference urine was calculated by dividing the found amounts ofnatSe, 77Se or 76Se by the injection volume.

Also, in order to evaluate the Se recovery in the column RP

C18, the total amount of selenium obtained in the urine samples

was calculated by the integration of all the Se chromatographic

peaks in the urine samples divided by the total Se amount

previously determined.12 Results showed recovery values of 96 �6% of Se in this column.

Application of HPLC-IPD-ICP-MS to determination of natural

and exogenous selenospecies in urine

The HPLC-IPD-ICP-MS validated quantitative methodology

was applied to determine the distribution of selenium (natural

and exogenous) species in the urine of the supplemented and non

supplemented groups. As previously explained, the chromato-

gram (signal vs. min), allowed species separation while by

applying IPD, the Mass flow chromatograms (mg Se vs. time

(min)) for both natural and exogenous selenium were obtained.

A typical mass flow chromatogram obtained for natSe in the

non supplemented group is showed in Fig. 5. As can be seen,

selenium turned out to occur mainly in the form of selenite

(retention time ¼ 2.7 min). Particularly the first day of the study

also a minor peak, at retention time of the selenosugar (natSe-

sugar) was detected. However this peak was hardly detected after

the second day of the study.

For comparison, Figs. 6-a and 6-b illustrate the mass flow

profiles obtained for ‘‘enriched’’ and ‘‘natural’’ abundance sele-

nospecies in the supplemented rats group, along the 14 days

supplementation period. In Fig. 6-a, the two major selenium

Fig. 5 Element Se distribution in urine (mg natSe/min vs time(min)


peaks at the retention times of 7.8 and 2.7 min should correspond

to natSe-sugar and natSelenite, respectively. However, Fig. 6-b

shows that 77Se profile at the 7.8 min peak increases steadily with

the supplementation time. Also, the amount of 77selenosugar

became higher than natSe-sugar after around 6 days. After 14 days

of supplementation the percentage of ‘‘exogenous’’ selenium

(77Se) in the selenosugar reached around 60% of total selenium

present in this compound. Fig. 6 show also that 77selenite was

detectable after 4–5 days of supplementation, but at levels lower

than those found for natSelenite (see Fig. 6-a for natSe).

The obtained cumulative urine excretion for endogenous and

exogenous selenium, from the supplemented group, is shown in

Fig. 7. Every data point corresponds to the average concentra-

tion of selenium excreted daily in urine by the three studied rats.

As can be observed, both total exogenous and natural selenium

increased linearly with time, but the observed cumulative amount

of 77selenium in urine was lower than that for natSe. Regarding

selenosugar excretion a linear increase during the period (slope ¼0.10, r ¼ 0.998) was observed, with similar slopes for natSe-sugar

and 77Se-sugar. However, after 11 days of supplementation the

cumulative amount of enriched selenosugar turned out to be

higher than that of the natural one.

Finally, the results obtained for inorganic selenium show that

the cumulative amount of natSe increases with a slope similar to

that of the selenosugars (slope ¼ 0.11, r¼ 0.998). Interestingly,

however, inorganic 77Se increased but at a substantially lower

slope (0.02, r¼ 0.996) and only after 7 days of supplementation.

According to previous basic publications on selenium metab-

olism8,20 it is well known that selenium absorbed by gastroin-

testinal tract passes to blood and is transported to the liver. In the

) for the non-supplemented group during the lactating period.


Fig. 6 a: natSe distribution in urine (mg natSe/min vs time(min)) for the supplemented group during the lactating period. b: 77Se distribution in urine (mg77Se/min vs time(min)) for the supplemented group during the lactating period.

Fig. 7 Amount of total and selenospecies containing endogenous or

exogenous selenium excreted in urine for the supplemented group with

respect to the total amount of selenium supplemented.


liver endogenous and exogenous selenium should be used for

selenoproteins synthesis which would pass into the bloodstream

and then be distributed to other tissues (brain, bone, muscle, etc).

Selenium not used in selenoprotein synthesis is eliminated by the

kidneys as selenosugar.21 However, in our study the presence of

selenosugar in urine of the non-supplemented rats was unde-

tectable (Fig. 5), suggesting that this particular metabolite

synthesis is related to selenium status or intake of the rat. In

other words, in the light of our results, levels of this selenosugar

in urine could be a best biomarker to assess selenium nutritional

status in the body (much more appropriate than the use of total

selenium values).

The inorganic selenium detected in urines, from the supple-

mented group (natural or exogenous) and from the non-supple-

mented group alike, might come from selenium incorporated

into actively regenerating proteins which can be then eliminated

during the degradation and then transformed to selenide for

reutilization or excretion.20


The fact that exogenous inorganic selenium is not found in

urines until the 5–6 day (Fig. 7), could indicate the slow turnover

of selenoproteins catabolism in the different body compartments

(brain, bones, testes,.).20 This assumption could be supported

by the fact that in the non-supplemented group virtually only

inorganic selenium was detected (Fig. 5) showing clearly the

endogenous Se metabolism differences between the non- and the

supplemented groups. That is, the ‘‘natural’’ inorganic selenium

found in urine could be related with the slow turnover of sele-

nium from tissues, whereas the found ‘‘natural’’ selenosugar

levels should be more related with Se reutilization into general

body proteins (Fig. 6-a). Conversely, the earliest peak of 77sele-

nosugar (Fig. 6-b) in urine would probably represent Se material

that is not utilised, whereas the 77selenite later peak could indicate

the part of supplemented Se involved in Se-proteins synthesis and

metabolism. In brief, most likely the selenosugar could be

a better Se nutritional marker than total selenium measurements.

Conclusions

The use of two stable isotopes (one as nutritional and the other as

quantification tracer) in combination with IPD-ICP-MS and

HPLC-ICP-MS with IPD, has allowed the quantification of

endogenous and exogenous total selenium and also of seleno-

species present in urine.

Apparent absorption of 77Se (i. e. supplements of Se) was not

significantly different during the study (with an average value of

98.8 � 0.5%). However, the apparent retention percentage was

about 84% the first day of supplementation, decreased gradually

and dropped sharply after the 11 day (at this time the enriched

selenium amount excreted in urine was higher than that of the

natural one, and the isotope ratio 77Se/natSe becomes similar in

urine and serum12).

Most excreted selenium was eliminated in urine and natural or

exogenous Se-sugar happened to be the main selenocompound

present in the supplemented group urines. Interestingly, this

metabolite was not present in the urine of rats fed with a non-

supplemented (seleno-deficient) diet. However, natural inorganic

selenium was found in both supplemented and non-supple-

mented rats (the latter one in lower amounts) whereas exogenous

inorganic selenium appeared only after 5 days of supplementa-

tion in the supplemented group. So, inorganic selenium detected

in urine possibly comes from selenium incorporated into actively

regenerating proteins which can be eliminated after their degra-

dation and transformed to selenide20 for neutralization or

excretion.

Conversely, the selenosugar seems to be a urinary metabolite

which represents the selenium not incorporated to selenoprotein

synthesis, being that excess transformed in sugar metabolites

(probably in order to overcome selenide excess toxicity).

Of course, quantitative data of seleno-proteins and

Se-metabolites in different fluids and tissues could be obtained in

the future by the proposed methodology to investigate and

clarify the biological significance and pathways of selenium as


a nutraceutical element. Also, the methodology proposed here

could provide invaluable quantitative speciation data to unveil

elusive Se metabolism pathways helping to design more robust Se

compartmental models.20

Acknowledgements

The authors are grateful to: ‘Fundaci�on para la Investigaci�on

Cientı́fica Aplicada y la Tecnologı́a, Principado de Asturias

(FICYT)’ for financial support through Project, reference PC-06-

007.

Additional financial support from Ministerio de Ciencia y

Tecnologı́a (Project reference CTQ2006-02309BQU), as well to

LABORATORIOS ORDESA (Barcelona, Spain) and

Fundaci�on Grupo Castrillo (Spain) is gratefully acknowledged.

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Enriched stable isotopes and isotope pattern deconvolution for quantitative speciation of endogenous...

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