X-ray fluorescence elemental mapping and microscopy to follow hepatic disposition of a Gd-based...

X-ray fluorescence elemental mapping and microscopy to follow hepaticdisposition of a Gd-based magnetic resonance imaging contrast agent

Riccarda Delfino,*† Matteo Altissimo,‡ Ralf Hendrik Menk,§ Roberto Alberti,{ Tomasz Klatka,{

Tommaso Frizzi,{** Antonio Longoni,{ Murielle Salome,†† Giuliana Tromba,§ Fulvia Arfelli,‡‡ Milan

Clai,§§ Lisa Vaccari,§ Vito Lorusso,{{ Claudio Tiribelli* and Lorella Pascolo§***

*Centre for Liver Studies, AREA Science Park, †Department of Pharmaceutical Sciences, University of Trieste, ‡‡Department of

Physics, University of Trieste and INFN, §Synchrotron Trieste S.C.p.A, §§University Hospital Trieste, ***Institute for Maternal

and Child Health, IRCCS Burlo Garofolo, Trieste, {Department of Electronics and Informatics, Politecnico of Milan and INFN,{{Bracco Imaging S.p.A, **XGLab s.r.l., Milan, Italy, ‡CSIRO Materials Science and Engineering, Melbourne, Australia and

††European Synchrotron Radiation Facility, Grenoble, France

SUMMARY

1. Spatially resolved X-ray fluorescence (XRF) spectroscopy

with synchrotron radiation is a technique that allows imaging

and quantification of chemical elements in biological specimens

with high sensitivity. In the present study, we applied XRF tech-

niques at a macro and micro level to carry out drug distribution

studies on ex vivomodels to confirm the hepatobiliary disposition

of the Gd-based magnetic resonance imaging contrast agent

B22956 ⁄1.

2. Gd presence was selectively quantified allowing the determi-

nation of the time dependent disappearance of the drug from

blood and its hepatic accumulation in mice after administration.

Elemental mapping highlighted the drug distribution differences

between healthy and diseased livers. XRF microanalyses showed

that in CCl4-induced hepatitis, B22956 ⁄1 has greatly reduced

hepatic accumulation, shown as a 20-fold reduction of Gd pres-

ence. Furthermore, a significant increase of Fe presence was

found in steatotic compared with healthy livers, in line with the

disease features.

3. The present results show that XRF might be useful in pre-

clinical pharmacological studies with drugs containing exogenous

elements. Furthermore, quantitative and high-sensitivity elemen-

tal mapping allows simultaneous detection of chemical variation,

showing pathological conditions. This approach was useful in

suggesting reduced B22956 ⁄1 accumulation in steatotic livers,

thus opening possible new diagnostic perspectives for this drug.

Key words: chemical imaging, liver, magnetic resonance

imaging contrast agents, X-ray fluorescence.

INTRODUCTION

X-ray fluorescence (XRF) spectroscopy (XRF) is a powerful tech-

nique for qualitative and quantitative chemical imaging in biological

specimens. Exploiting the penetrating capability of high-brilliance

X-ray synchrotron radiation micro-beams, attogram sensitivity in bio-

logical samples has been achieved in microscopy for some transition

metals and other biologically relevant trace elements.1 With labora-

tory instrumentation based on conventional X-ray generators coupled

to polycapillary lenses, a spatial resolution of the order of some tens

of micrometers and a sensitivity of the order of 10 ppm can be

reached for selected elements.2,3 Spatial resolution down to 100 nm

can be achieved routinely in micromapping applications with third-

generation synchrotron light sources. With the next-generation

microprobes under development, a resolution limit of < 30 nm will

be achieved.4–6

The possibility of focusing an incident monochromatic X-ray

beam generated by a synchrotron source enables spatially-resolved

XRF microspectroscopic imaging. This allows simultaneous specific

chemical mapping for different elements of interest. Its high spatial

resolution and large penetration depth makes synchrotron Radiation

XRF (SR-XRF) one of the few techniques capable of quantitative

in situ elemental imaging over a range of length scales, from the

cellular level to micrometric histological sections.7

In the present work, we describe the application of XRF in the

study of pharmacological biodistribution. In particular, synchrotron

radiation (SR) XRF microspectroscopy was used to determine the

hepatic disposition of the magnetic resonance imaging (MRI)

contrast agent B22956 ⁄1, by taking advantage of the presence of the

exogenous element gadolinium (Gd).

Gadocoletic acid trisodium salt, or B22956 ⁄1, is a new Gd-based

magnetic resonance (MR) contrast agent that has been formulated to

be used as a blood pool agent, as its structure assures high binding to

plasma proteins.8 Pharmacological studies in animal models and in

healthy human volunteers have shown that biliary excretion is the

primary route of elimination of the contrastographic B22956 ⁄1,9

which is mainly mediated by the canalicular adensosine triphosphate

(ATP)-dependent transporter, multidrug resistance-related protein 2

(MRP2; ABCC2). Additionally, we recently reported that the

accumulation of this compound in human hepatic cells is mediated

Correspondence: Dr Lorella Pascolo, Institute for Maternal and Child

Health, IRCCS Burlo Garofolo, Via dell’Istria 65 ⁄ 1, 34137 Trieste, Italy.

Email: [email protected]

Received 25 October 2010; revision 23 September 2011; accepted 26

September 2011.

� 2011 The Authors

Clinical and Experimental Pharmacology and Physiology

� 2011 Blackwell Publishing Asia Pty Ltd

Clinical and Experimental Pharmacology and Physiology (2011) 38, 834–845 doi: 10.1111/j.1440-1681.2011.05618.x

by liver-specific transport proteins (organic anion transporting poly-

peptide (OATP) transporters, particularly OATP1B3),10 and thus is

potentially affected in those diseases where the expression of these

transporters is changed.11 In animal models, it was reported that is

chaemia in mice produces a marked reduction in the expression of

both basolateral passive transporters (including oatps) and canalicular

export pumps (including Mrp2).12 In rodents, liver dysfunction

induced by CCl4 administration causes altered hepatic elimination of

different drugs, in particular reduced biliary excretion of organic

anions, and seems to be partially related to changed expression of

organic anion transporters.13,14 With regard to MRI contrast agent

categories, which are mainly organic anions, it was reported that the

contrast agent Gd-BOPTA has decreased biliary excretion and modi-

fied hepatic distribution in rats with final stage biliary cirrhosis.9,15

Similarly, in clinical practice with Gd-BOPTA, an altered signal

enhancement has been reported in some benign hepatic diseases (as

focal fatty infiltration), potentially in concurrence with altered accu-

mulation of the contrast agent in hepatocytes.16 Magnetic resonance

imaging is highly effective in detecting hepatic anatomical dysfunc-

tions and neoplastic lesions, especially when Gd-based contrast

agents are used. However, the limited spatial resolution of MRI does

not allow determination of whether these commercial Gd complexes

are really accumulated differently in healthy and diseased hepato-

cytes. Solving this issue would be of particular diagnostic interest if it

shows specific change in hepatic disposition during the early stages

of both benign and malign diseases. In the present study, we suggest

the use of XRF imaging at two length scales to clarify the major

features of Gd complex distribution in mouse liver.

We report on the time-dependent hepatic disposition of B22956 ⁄ 1

in mice, and the different accumulation in healthy and CCl4-treated

animals. SR-XRF microscopy on histological slices is also described,

that correlates the contrast agent distribution and some endogenous

element changes with the pathological condition. The possibility of

imaging Gd concentration with high spatial resolution at the intracel-

lular level in tissue is reported.

METHODS

Chemicals and materials

Gadocoletic acid trisodium salt (Bracco code B22956 ⁄ 1), which is a trisodium

salt derivative of gadopentetate, was synthesized by methods described previ-

ously.15 B22956 ⁄ 1 was pharmaceutically formulated as sterile and apyrogenic

0.25 mol ⁄L (39.25 mg ⁄mL of gadolinium) aqueous solution for injections

without additional eccipients. Different dilutions in water of the B22956 ⁄ 1

stock solutions were prepared in order to achieve a final concentration ranging

from 10 mg ⁄mL to approximately 1 lg ⁄mL to be used for quantitative cali-

bration of XRF measurements.

500-nm thick silicon nitride membranes, with an area of 1 mm2, fabricated

by means of a standard combination of ultraviolet lithography, dry and wet

etching, were kindly provided by the LILIT group (TASC-INFM Nanolitho-

graphy beamline at the Elettra). All other chemicals, unless otherwise speci-

fied, were purchased from Sigma-Aldrich (Milan, Italy).

Animal studies

Female CBA mice weighing 20–25 g were used for the present study. Ani-

mals were housed in standard plastic cages at a constant temperature of 22�C,

with a 12-h light-dark cycle and ad libitum access to water and standard

mouse diet. The colony was maintained specific-pathogen free in the animal

house of the University of Trieste. All the experimental procedures were

carried out in accordance with the Animal Care and Use guidelines of the eth-

ics Committee of The University of Trieste.

To carry out pharmacokinetic studies, six animals were given 0.2 mg ⁄ g of

B22956 ⁄ 1 (0.189 lmol ⁄ g bodyweight) by injection in the tail vein. At 5, 15

and 30 min after the injection, two mice were killed by cervical dislocation

and 200 lL of venous blood were collected into heparinized tubes. At the

same times, the liver and other organs were explanted rapidly from carcasses,

rinsed extensively with PBS, immediately frozen in M-1 embedding medium

(Thermo Shandon, Pittsburgh, PA, USA) and stored at )20�C until section-

ing. The experiment was carried out in duplicate.

To induce steatosis in mice, CCl4 administration was selected as a well-

characterized and widely available model of hepatocellular injury.17 Three

animals were given 1 lL ⁄ g of CCl4 dissolved in corn oil i.p. whereas control

animals received an equal volume of corn oil only (100 lL). All animals

treated with CCl4 developed steatosis.

A total of 24 h later, the animals were given 0.2 mg ⁄ g of B22956 ⁄ 1 by

injection in the tail vein, as described before. At 15 min after the injection, the

mice were killed and samples were collected. The tissue samples analysed at

Elettra were prepared by removing the excess of embedding medium and slic-

ing the sample with a cryostat at )18�C ⁄)20�C (Leica Microsystems GmbH,

Wetzlar, Germany). The samples were cut to dimensions of 0.9 cm · 0.9

cm · 0.5 cm in order to be housed in an appropriate plastic support of exter-

nal dimensions 1 cm · 1 cm · 0.5 cm. As a result of the particular morpho-

logy of mouse liver, the left lobe was selected for quantitative measurement,

allowing a better estimate of the overall organ distribution, whereas the centro-

lobular portion including the gallbladder was preferred for XRF mapping in

order to monitor possible biliary excretion.

Then, 5-lm sections of frozen liver tissue were mounted on silicon nitride

windows and air-dried. Each specimen was mounted into the ID-21 Scanning

X-ray Microscope vacuum chamber, at the European Synchrotron Radiation

Facility (ESRF), Grenoble (France).

Histological examination

For the histological analysis, 5-lm sections of the frozen liver tissue were

mounted on glass slices and air-dried. Samples were stained after standard

procedures utilizing haematoxylin–eosin solution for cryostatic sections and

analysed using a Leica microscope (Leica Microsystems GmbH).

Description of XRF experimental set-up at SYRMEP (Elettra)

At the SYRMEP beamline of the Elettra synchrotron light source in Trieste

(Italy),18 XRF spectra were obtained by utilizing a collimated

(500 lm · 500 lm for element mapping) monochromatic X-ray beam with

an incident photon energy of 9 keV.

A compact detection module equipped with a high-performance X-ray sen-

sor was developed by the authors. This includes a new 10-mm2 ‘droplet type’

SiliconDrift Detector (SDD) cooled by a Peltier element to a temperature lower

than 0�C.2,19 The fluorescence detection system has a spectroscopic resolution

of 133 eV (FWHM) at the Mn Ka line (5.89 keV) and a peak-to-background

ratio of approximately 800.3 Its sensitivity allows the identification of chemical

elements with atomic numbers above Z = 11 (Na) and the presence of trace

elements down to approximately 1 ppm. In order to minimize the Compton

background, the detector was mounted perpendicular to the incident beamwith

a detector-to-sample distance of 3 cm. A charge-coupled device camera down-

stream of the sample was used to monitor the beam-sample alignment

(Fig. S1a, in supplementary material). A custom developed readout, control

and acquisition unit,4 specially designed for elemental mapping applications,

allowed automatic mesh scans of selected areas with online data visualization

and other features, such as energy calibration and emission peak identification.

Tissue and blood XRF quantification, XRF tissue mapping

In order to quantify Gd concentrations, the system at the SYRMEP beamline

was calibrated using different solutions of B22956 ⁄ 1 ranging from

6.73 lg ⁄mL (6.37 lmol ⁄L) to 67.3 mg ⁄mL (63.7 mmol ⁄L), which translates


Clinical and Experimental Pharmacology and Physiology � 2011 Blackwell Publishing Asia Pty Ltd

XRF imaging and Gd-B22950 agent 835

into Gd concentrations ranging from 1 lg ⁄mL to 10 mg ⁄mL. The calibration

solutions, as well as the blood samples, were enclosed in plastic test tubes and

were analysed using a collimated, monochromatic X-ray beam at a photon

energy of 9 keV and with a spatial extension of 8 · 2 mm at the sample posi-

tion. The beam energy was chosen to be slightly above the L12s-edge of Gd

(8.376 keV) in order to optimize the excitation efficiency for this element.19

Blood samples were analysed using the same geometry as that used for the

system calibration. For quantitative measurements, liver samples were also

analysed using a similar geometry.

During imaging, liver samples accommodated in their plastic supports

(1 · 1 · 0.5 cm) were installed on an XY motorized translation stage con-

trolled by the computer that also controlled the measurement procedure and

data acquisition. The samples were enclosed in a semi-open hood flooded with

dry nitrogen and cooled by a thermoelectric Peltier element. In this case, the

9 keV X-ray excitation beam was collimated down to 500 · 500 lm at

the sample position. Typically, the samples were raster scanned using a

pitch of 500 lm in both directions and covering an area of 10 · 10 cm. The

dwell time per point ranged from 30 to 60 s.

Micro XRF elemental analysis

The Micro XRF element analyses were carried out at the ID 21 beamline of

the ESRF using a set-up that has been described previously.20 A monochro-

matic X-ray beam was focused onto the sample. The photon energy of the

X-rays was set to 7.3 keV, which is the highest energy available at this beam-

line. The spot size was either 2 lm · 2 lm or 500 nm · 500 nm. The sample

was mounted on a custom XYZ stage, tilted by 30� from the optical axis,

facing a Ge fluorescence detector placed at 3.5 cm from the sample. A photo-

diode was placed downstream of the sample, slightly off axis. This allowed

the collection of transmission data, while simultaneously collecting the fluo-

rescence signal, as described previously.21,22 Fig. S1b in the supplementary

material shows the experimental set-up.

Images were acquired by raster scanning the samples through the stationary

beam. The step size ranged from 0.5 to 2 lm and the dwell time was

2 s ⁄ pixel. The fluorescence images were then processed with the PyMCA

software, which allows the extraction of qualitative and quantitative data. Rel-

ative quantifications are obtained from internal software calibration, setting

sample (composition and thickness) and measuring parameters,23 as we previ-

ously reported.24

Software

At Elettra, data acquisition was carried out using the PoliMiMCA software

developed by Politecnico di Milano (Milan, Italy) and Matlab (The Math-

Works, Natick, MA, USA) was used for data processing.

At ESRF, the PyMCA software23 developed by the Beamline Instrumenta-

tion Software Support group (BLISS) of the ESRF was used for micro XRF

spectra acquisition and analysis.

Statistical analysis

Results are expressed as the mean ± SD (standard deviation) of three assays

(two in one case) per condition. Statistical analysis was carried out using anal-

ysis of variance combined with Student’s two-tailed t-test. Differences among

the conditions were considered significant at P < 0.05. Correlations between

elemental maps were evaluated using Pearson’s linear correlation coefficient,

which shows the strength of a linear relationship between two variables. The

correlation coefficient, R, is a scalar quantity in the interval ()1.0, 1.0) and is

defined as the ratio of the covariance of the sample populations to the product

of their standard deviations. It is a direct measure of how well two sample

populations vary jointly. A value of R close to +1 or )1 shows a high degree

of correlation and a good fit to a positive or negative linear model. A value of

R close to 0 shows a poor fit to a linear model. In order to describe the homo-

geneity of an elemental map, the relative error rR defined as the mean value

of the image photon counts divided by the standard deviation has been intro-

duced, which can be compared with the relative error as a result of Poisson

(photon) statistics rP. A map with (rP ⁄rR)2 > 40% is considered homo-

geneous.

RESULTS

Quantitative kinetics of B22956 ⁄1 in blood, liver and tissues of

healthy mice by XRF means

The system calibration for the data acquired at Elettra (Fig. S2)

shows that under the described experimental conditions and geome-

try, the minimum detection limit is 5 lg ⁄mL of Gd, corresponding to

33.63 lg ⁄mL (31.85 lmol ⁄L) of contrast agent. The applied condi-

tions allowed analysis of both blood samples and frozen tissue

samples.

The pharmacokinetic distribution curves were determined by col-

lecting mouse liver and blood samples at 5, 15 and 30 min after i.v.

injection of a single dose of B22956 ⁄1 (0.189 lmol ⁄g). Different

experiments were carried out in at least three periods of beamtime

scheduled at Elettra, which gave comparable results. In Fig. 1, we

report the results for the distribution curves of B22956 ⁄1 in the liver

(Fig. 1a) and in blood (Fig. 1b) from healthy mice. It can be seen that

the Fe signal in the blood spectra is as prominent as expected (Fe Ka

at 6.4 keV and Fe Kb at 7.06 keV) and partially overlapping the Gd

L peaks. In the liver, the Fe signal is lower whereas the Gd peak

remained almost the same.

Only the Gd concentration can be quantified. Other elemental vari-

ations are only qualitative, as they have not been specifically cali-

brated. The pharmacokinetic trend reported in Fig. 1c shows a rapid

disappearance in blood, where the first registered B22956 ⁄1 concen-

tration (5 min) is approximately 583.4 lmol ⁄L, decreasing to 83%

(485.7 lmol ⁄L) and 25% (146.5 lmol ⁄L) at 15 and 30 min respec-

tively. In agreement with the main biliary elimination of B22956 ⁄ 1

already reported, a rapid and time-dependent accumulation in the

liver was registered; the XRF measurements showed concentrations

of 305.7 lmol ⁄L, 790.4 lmol ⁄L and 785.3 lmol ⁄L at 5, 15 and

30 min, respectively.

XRF analyses on spleen and kidney samples of the animals given

the contrast agent (data not shown) showed undetectable or negligi-

ble presence of Gd.

Animal model of hepatitis (early steatosis)

The induction of liver steatosis by CCl4 treatment was verified by

both histological examination of liver tissue and assessment of serum

enzyme activities. As shown in Fig. 2, compared with the control

(Fig. 2a), the liver of a CCl4-treated mouse (Fig. 2b) presents marked

signs of steatosis and initial necrotic processes. The hepatocytes of

steatotic tissue are increased in size with the presence of many lipid

vacuoles in the cytoplasm. In agreement with a poisoning hepatitis,

necrotic cells are widely spread within the tissue and are not limited

to the perivascular area.

Serum enzymes for hepatobilary disease confirmed the pathologi-

cal model for the CCl4-treated mice; aspartate aminotransferase

(AST) and alanine aminotransferase (ALT) activities have values that

are 100-fold (22 624 vs 223 UI ⁄mL) and 464-fold (23 672 vs

51 UI ⁄mL) the normal upper limit. No significant variation was

found for cGT (1 vs 0 UI ⁄mL, CCl4-treated vs normal mouse).

Figure 1d shows blood spectra acquired in healthy and steatotic

animals at 15 min after B22956 ⁄ 1 was given. In line with the



836 R Delfino et al.

reduced hepatic function, a higher level of Gd was detected in the

blood of steatotic animals, showing prolonged presence of B22956 ⁄ 1

and decelerated excretion.

Elemental mapping in healthy and steatotic mouse livers

In Fig. 3, the elemental analysis of a healthy liver specimen is shown.

The leftmost upper panel shows an optical image of the tissue sample

containing the gallbladder region inserted in a plastic frame. The ele-

mental distribution profile is obtained as a result of a deconvolution

of the fluorescence spectra. Because it was not possible to obtain

calibrated concentrations for these element maps, the pixel values of

the latter were normalized to the maximum of the K map using the

following algorithm, where m(x,y) and p(x,y) denote the measured

and the normalized pixel value of an elemental map at the position

x,y, respectively:

(b)

(a)

Fig. 2 Steatotic model: histological analyses and blood distribution. Histo-

logical analyses of 5-lm frozen sections of (a) steatotic and (b) healthy mouse

liver. Slices were stained with haematoxylin–eosin solution for cryostatic

sections and analysed using a Leica microscope (magnification: · 100).

(a)

(b)

(c)

(d)

Fig. 1 Kinetics of distribution for B22957 ⁄ 1 in mouse blood and organs. (a)

The liver spectra acquired at 5 (black), 15 (red) and 30 m (green) after the i.v.

injection of 0.189 lmol ⁄ g bodyweight of B22956 ⁄ 1. X-ray beam energy 9

keV, spot size approximately 500 · 500 lm. (b) Spectra acquired from blood

samples at the same times. X-ray excitation 9 keV, spot size approximately

8 · 2 mm. (c) Pharmacokinetic trend of B22957 ⁄ 1 in blood (¤) and liver

(•) according to the calibrated Gd content. The means and standard deviation

of six animals (three measurements each) are shown. (d) The blood spectra of

healthy (black) and steatotic (grey) mice. Blood was collected 15 min after

i.v. injection of 0.189 mmol ⁄ kg of B22956 ⁄ 1. X-ray beam 9 keV, spot size

approximately 500 · 500 lm.

b




pðx; yÞ ¼ maxðKÞ �mðx; yÞ �minðmÞ

maxðmÞ �minðmÞ

Furthermore, max(K) is the maximum value found in the K map

and min(m) and max(m) are the minimum ⁄maximum values found

in the respective elemental map.

In healthy livers, a clear co-localization of S, Gd, K and Ca in the

gallbladder portion was found, which could be statistically proven by

the correlation coefficient R between the different element popula-

tions in this region of interest. The following correlations were found:

Gd � Ca (R = 0.721), Gd � K (R = 0.762) and Gd � S

(R = 0.885). It can be seen that Fe is very low Gd � Fe (R = 0.042)

in this area, whereas it is quite homogeneously distributed in the

healthy liver tissue (rR = 0.06, rR being the relative error defined as

the mean value of the image values divided by the standard deviation.

The relative error as a result of Poisson (photon) statistics is

rP = 0.04). The high concentration of Gd in the gallbladder shows

the presence of B22956 ⁄1 and is clearly related to the biliary elimina-

tion process of the drug. The intensity of the signal is strongly related

to tissue thickness (as verified by X-ray beam absorption analyses),

whereas concomitant high concentrations of Fe and Gd in the same

zones seem related to hidden vessels and ⁄or blood stagnation.

Because samples were prepared by rapid freezing of excised organs,

residual drug in the blood could affect, to a certain extent, the quanti-

tative measures and maps. This is particularly true in the central

region of the organ, where many vessels are present. Some abnormal-

ities in the K (and Cl) distribution, which extended over tissue bor-

ders, are a result of the presence of K and Cl containing embedding

medium used in the sample preparation.

When elemental analyses are carried out in the steatotic mouse

liver (Fig. 4), the mapping, which is the result of a deconvolution of

fluorescence spectra profile, appears less homogeneous as expected

for an injured tissue (rR = 0.21 instead of rR = 0.06) and high

intensity areas appear for elements, such as Ca, K, and Fe. The corre-

lation coefficients, R, between those maps and the Gd map are

Gd � Ca (R = 0.789), Gd � K (R = 0.772), Gd � Fe (R = 0.740).

Only for Gd � S (R = 0.03) do the populations seem to be less corre-

lated. The gallbladder was included in this sample (Fig. 4a).

(a) (b) (c)

(d) (e) (f)

Fig. 3 Macro-elemental mapping in healthy mouse liver 15 min after B22956 ⁄ 1 was given. Synchrotron radiation X-ray fluorescence elemental scan, 9.0 keV

X-ray beam energy, frozen section of healthy mouse liver at 15 min after B22956 ⁄ 1 was given. (a) Optical image of a central portion of the organ, 50–100 lm

below the scanned area (cryostatic excision) and exposing the gallbladder region (1 · 1 · 0.5 cm). Same sample, single element maps of (b) iron, (c) calcium, (d)

potassium, (e) gadolinium and (f) sulphur. Scanned area: 9 · 9 mm, 500 lm pitch, acquisition time 60 s ⁄ pixel. The colour table is the relative count intensity

scaled to the maximum intensity of the K map. Bar, 2 mm for all images.




As expected, K, S and Gd co-localize in the gallbladder area, where

the high concentration of Ca could be indicative of some biliary

excretion impairment.

Micro XRF elemental analysis in histological mouse liver

specimens

Quantitative micro XRF microscopy (under vacuum) was carried out

on liver slices from healthy and steatotic mice after treatment with

B22956 ⁄ 1. The microscope imaging end-station at ID21 of the ESRF

allows optical imaging at a micrometric level, before and during the

XRF measurements. As shown in Figs 5a,6a, images of the analysed

regions were also acquired using an external visible light microscope.

Elemental mapping was obtained by deconvolving the X-ray fluores-

cent signals with PyMca software.

Fig. 5 shows the elemental mapping of P, S, Cl, K, Ca, Gd and Fe

in a 100 · 100-lm section of the liver sample from a healthy mouse

15 min after administration of B22956 ⁄1. The sample analysed is a

reduced slice of a specimen considered in the pharmacokinetic curve

of Fig. 1. Elemental mapping shows that all the elements are concen-

trated in the hepatic cells. Interestingly, the high signals of Gd associ-

ate with high P (R = 0.762 for Gd � P) and S (R = 0.739 for Gd

� S) signals, suggesting that the Gd-based drug uptake depends on

cell density in the tissue.

In Fig. 6, microscopic XRF analyses are shown in a liver sample

from a CCL4-treated mouse 15 min after administration of

B22956 ⁄1. Compared with the data in Fig. 5, the steatotic tissue

shows a clearly decreased concentration of Gd with respect to P and

S abundance (R = 0.601 for Gd � P and R = 0.511 for Gd � S).

To better appreciate variations in elemental concentrations, differ-

ent regions of interest of the liver specimen sections reported in

Fig. 5 (two areas of a single tissue) and Fig. 6 (three areas of a single

tissue) with the same dimension of 25 · 25 lm2 were analysed by

the MAPS software and compared in terms of total mass fraction

concentration of the different elements (Table 1). Quoted are the

mean values of the counts within these regions of interest, which are

proportional to the element concentration. Subsequently, the standard

error was calculated as the square root of the variance from the mean.

A theoretical matrix composition was considered to normalize

and calibrate signal intensities and thus obtain a semi-quantitative

(a) (b) (c)

(d) (e) (f)

Fig. 4 Macro-elemental mapping in steatotic mouse liver 15 min after B22956 ⁄ 1 was given. Synchrotron radiation X-ray fluorescence elemental scan elemental

scan, 9.0 keV X-ray energy, frozen section of steatotic mouse liver 15 min after B22956 ⁄ 1 was given. (a) Optical image 50–100 lm below the mapped area

(cryostatic excision) highlighting inclusion of the gallbladder region. The sample is not inserted into the plastic frame. Single element maps for (b) iron, (c) cal-

cium, (d) potassium, (e) gadolinium and (f) sulphur. Scanned area: 9 · 9mm, 500 lm pitch, acquisition time 60 s ⁄ pixel. The colour table is the relative count

intensity scaled to the maximum intensity of K map. Bar, 2 mm for all images.




evaluation of the elements.24 As shown in Table 1, P, Ca, Fe and Gd

are the elements with the highest changes when comparing average

values in healthy and steatotic tissues. Although there is apparently

no change in the S content in the steatotic versus healthy sample

(P > 0.1), there is a trend towards a decrease in the P ⁄S ratio

(1.21 ± 0.21 vs 1.52 ± 0.14, P < 0.05), which is an indicator of cel-

lular state. From average data, Gd presence in the steatotic liver is

estimated to be 95% lower compared with normal liver areas. The

concurrent increase of Fe presence (35%) and the decrease of Ca

(30%) are also significant.

The content of P in biological samples, as a constituent of non-

diffusible solutes, nucleic acids and cell membranes, has been

previously correlated with cell mass and density in studies using

X-ray microanalyses.25 The apparent decrease could partially reflect

the extensive structural damage in steatotic tissue. Similar calcula-

tions have been carried out on remaining tissue samples (steatotic vs

control) from all animals (two steatotic and two healthy animals at

15 min after administration). Similar variations of the Gd (85–95%

reduction in steatotic vs normal) and Fe (30–40% increase in steatotic

vs normal) content were estimated. Because results were obtained

during different beamtimes with slightly different experimental set-

ups (detection efficiency and beam intensity), results are not very

comparable in terms of total counts and are therefore not grouped in

a single table.

Figure 7 shows micro XRF results at high spatial resolution

(0.5 lm pixel size) of a section of health liver 10 min after B-22956

administration. The X-ray absorption image (Fig. 7a) shows density

variations in the section compatible with cellular matter and intersti-

tial spaces. Potassium, being the most abundant intracellular cation,

delineates the intracellular spaces in the tissue section. The gadolin-

ium map (Fig. 7c), particularly when compared with K distribution,

shows that the element is quite exclusively intracellular (Fig. 7h).

Some signal increases in the K map tend to co-localize, to a certain

extent, with P, and to a lesser extent with other elements, possibly

showing specific intracellular regions of higher density. The other

analysed elemental concentrations, although clearly higher intracellu-

larly, are less specifically localized compared with Gd and K.

DISCUSSION

The present study shows the applicability of XRF techniques for

pharmacological investigations in ex vivo animals as a complemen-

tary method to other analytical and imaging techniques. The com-

pound in the present study is in fact a contrast agent (CA) for one of

the most powerful clinical imaging modalities (MRI). However, MRI

cannot reach the quantitative and spatial sensitivity we obtained with

XRF elemental analyses over the large range of scaling dimensions.

Gd-based MRI CA are complexes with very high stability con-

stants. On administration of pharmacological doses, they are not

metabolized and Gd release from the molecule is prevented during

the normal distribution and elimination times. This has been proven

for the drug used in the present study, which has high hepatic

(a) (b) (c)

(e) (f) (g)

(d)

(h)

Fig. 5 X-ray fluorescence microscopy of healthy mouse liver 15 min after B22956 ⁄ 1. Synchrotron radiation X-ray fluorescence elemental scan of the healthy

liver sample; 7.3 keV X-ray energy, slice thickness: 5 lm attached to a 500-nm thick silicon nitride membrane. (a) Optical microscope image reporting the

selected area. Single element maps for (b) phosphorus, (c) sulphur, (d) chloride, (e) potassium, (f) calcium, (g) gadolinium and (h) iron. The scanned area:

100 · 100 lm, step size: 2 lm, acquisition time: 2 s ⁄ pixel.




tropism, making it applicable for hepatobiliary diagnosis.26 Our data

show that the B22956 ⁄1 distribution in blood and organs can be fol-

lowed at different length scales by using the Gd X-ray fluorescent

signal. With an appropriate apparatus (Fig. S1a) and a proper calibra-

tion (Fig. S2), we carried out both quantitative analyses at adequate

sensitivity to show drug presence in blood and tissue samples, as well

as 2-D chemical imaging.

The quantitative pharmacokinetic data obtained (Fig. 1) are in line

with what is expected from previous studies on B22956 ⁄1 distribu-

tion in rodents.27 The hepatic disposition of B22956 ⁄ 1 in mice is

clearly shown by measuring increasing concentrations in liver over

the time after administration and the concurrent reduced presence in

blood (Fig. 1c). The most interesting results obtained with the macro-

scopic imaging are the two-bidimensional elemental maps showing

the Gd presence in the gallbladder region of both healthy (Fig. 3) and

steatotic (Fig. 4) livers. This confirms the active biliary elimination

of the MRI contrast agent during normal and reduced hepatic func-

tion. In both gallbladders, the high presence of S detected could be

related to metabolites as sulphoconjugates (from bile acids and

organic anions) or thiolic compounds, also oxidized and reduced glu-

tathione. The high Ca concentrations in the bile-related region

(Fig. 4), as well as the inhomogeneity of some endogenous elements,

would encourage further investigations to search for specific

functional correlations. The apparatus we developed has a spatial

(a) (b) (c)

(e) (f) (g)

(d)

(h)

Fig. 6 X-ray fluorescence microscopy of steatotic mouse liver 15 min after B22956 ⁄ 1. Synchrotron radiation X-ray fluorescence elemental scan of the steatotic

liver sample; 7.3 keV X-ray energy, slice thickness: 5 lm attached to a 500-nm thick silicon nitride membrane. (a) Optical microscope image reporting the

selected area. Single element maps for (b) phosphorus, (c) sulphur, (d) chloride, (e) potassium, (f) calcium, (g) gadolinium and (h) iron. The scanned area:

100 · 100 lm, step size: 2 lm, acquisition time: 2 s ⁄ pixel.

Table 1 Element quantification in healthy and steatotic liver samples under synchrotron radiation X-ray fluorescence microscopy

Element Normal liver 15 mmmass

fraction (g ⁄ g)

Steatotic liver 15 mmmass

fraction (g ⁄ g)

Element variation

steatotic vs normal (%)

P 4.74 · 10)03 ± 1.74 · 10)04 3.83 · 10)03 ± 4.48 · 10)04 80.8 ± 4.2*

S 3.12 · 10)03 ± 6.01 · 10)05 3.16 · 10)03 ± 2.91 · 10)04 NS

Cl 1.45 · 10)03 ± 8.41 · 10)05 1.56 · 10)03 ± 1.1 · 10)04 NS

K 3.01 · 10)03 ± 9.54 · 10)05 2.83 · 10)03 ± 1.57 · 10)04 NS

Ca 4.24 · 10)05 ± 1.66 · 10)06 2.78 · 10)05 ± 4.3 · 10)06 65.6 ± 6.1*

Fe 1.10 · 10)04 ± 1.32 · 10)05 1.48 · 10)04 ± 1.46 · 10)05 134.6 ± 13.1**

Gd 2.71 · 10)04 ± 1.74 · 10)05 1.22 · 10)05 ± 4.8 · 10)06 0.45 ± 0.1**

Average values from 25 · 25 lm scanned areas and expressed as mass fraction (gram of element ⁄ gram of tissue). *P < 0.05; **P < 0.01.

NS, not significant.




sensitivity that allows testing of centimetre-sized samples, thus being

useful in pharmacological studies to rapidly select organs for further

histological analyses. Further improvement will be considered in

future for sample preparation for this set-up. Although the kinetic

data were in good agreement with the expected behaviour of the

drug, during map analyses we noted that a variable amount of resid-

ual blood in the frozen fixed organs could affect the results to a cer-

tain extent. As reported by other authors (Todd et al.),28 for instance,

the use of blood fixation procedures before tissue sampling could be

of particular interest in future analyses for avoiding effects of sudden

blood pressure variations, such as massive release of cellular metabo-

lites or contrast clearance, before imaging. The possible effect of

sample preparation procedure (such as the presence of residual blood

in the organ) should have more impact in the central part of rodent

livers, because in this region vessels of variable dimension are more

abundant. Kinetic analyses (as well as microXRF) were carried out in

portions of the left lobe of the organ, in order to avoid these artifacts.

At the same time, this choice allowed us to have a more reliable esti-

mation of the overall drug distribution and organ functionality.

When samples were analysed using microscopic imaging, the

quantification of elements at high spatial resolution increased func-

tional information for our models. A uniform distribution of endo-

genous elements in the healthy liver sample (Fig. 5) and the

simultaneous high-intensity signal for Gd clearly show that

B22956 ⁄1 is uniformly distributed in the hepatic tissue. At the high

spatial resolution shown in Fig. 7, a clear intracellular localization is

evinced for the contrast agent by comparing Gd maps with absorp-

tion images, and particularly with K localization (Fig. 7). Micro

elemental imaging of the diseased liver sample (Fig. 5) showed that

a steatotic condition impairs hepatic disposition of B22956 ⁄1 and

this seems to be related to a reduced hepatocellular accumulation.

By applying an algorithm for the quantification of Gd in cell

samples,24 a 90–95% reduction was in fact registered for Gd

presence in the steatotic compared with the healthy control at the

(a) (b) (c)

(e) (f)

(i) (j)

(g)

(d)

(h)

20 µm

Fig. 7 X-ray fluorescence microscopy of healthy mouse liver 10 min after B22956 ⁄ 1. Synchrotron radiation X-ray fluorescence elemental scan of a normal liver

sample 10 min after contrast agent was given; 7.3 keV X-ray energy, slice thickness: 5 lm attached to a 500-nm thick silicon nitride membrane. (a) X-ray absorp-

tion image. Step size: 500 nm, dwell time: 0.5 s ⁄ pixel. Single element maps for (b) potassium, (c) gadolinium, (d) calcium. (e) phosphorus, (f) iron and (g) sul-

phur. A co-localization for (h) K (green) and Gd (red) signals, (j) S (green) and Gd (red), and (i) P (green) and K (red) are shown. Scanned area: 50 · 50 lm, step

size: 500 nm, dwell time: 4 s ⁄ pixel.




same administration time. Specific reasons for reduced hepatic accu-

mulation are not shown by the present work. They could be con-

nected to a reduced blood supply caused by spreading necrosis

and ⁄or other factors prejudicing normal cellular functions, but this

needs further investigation. An important observation from the results

obtained is that the greatly reduced accumulation of the diagnostic

CA is associated with specific chemical changes in the hepatic tissues

that are symptomatic of cellular malfunction and tissue damage. The

data are particularly relevant when a possible clinical diagnostic

application of B22956 ⁄1 in MRI is considered to highlight morpho-

logical and functional changes of liver under diseased conditions.

The present technical approach could in fact be useful in an expanded

study for trying to associate the concentration of contrast agent accu-

mulation into the hepatocytes with the different early and late states

of cell sufferance.

CCl4 is a chemical that causes strong oxidative conditions and aci-

dosis in hepatocytes and some necrosis, as shown by histological

examination.17 A pH of approximately seven in hepatocytes was

reported in the case of CCl4 administration to rats.29 It is interesting

to note that some elemental variations we found by comparing nor-

mal and diseased liver (Table 1) could be explained by both oxida-

tive stress, acidosis and some cell necrosis. The S ⁄P ratio is generally

considered to reflect cell state, reflecting cell apoptosis progression

and is affected by extensive structural changes.30,31 A possible reduc-

tion in phosphorus spatial distribution could also be related to hepato-

cyte enlargement with increased presence of fat vesicles (Fig. 2) or a

reduced phosphate presence related to acidosis compensation. The

present results seem also to suggest a reduction of Ca in steatotic

liver, which is evident when comparing average quantifications. Fur-

ther investigations are needed to verify if this change can be related

to pH variations and oxidative stress mechanisms of diseased liver.

In Table 1, we also report that liver steatosis, when induced by

poisoning, causes a large Fe accumulation in hepatic tissue. This has

already been reported by other authors using a different approach.32

Thus, the present results confirm the previous evidence suggesting

the potential for using XRF microscopy for quantitative assessment

of the phenomena in experimental models or biopsies.

Chemical imaging represents an important challenge in biological

studies, as in principle, it could allow investigations of tissue damage,

cellular homeostasis parameters and cycle checkpoints, as well as dis-

turbed regulations. For instance, XRF has recently been used to eluci-

date the role of Fe, Zn or Co in neurons during neurodegenerative

diseases.33,34 The same trace elements were investigated in the liver

to characterize colorectal liver metastases over surrounding normal

livers.35 It should also be noted that gadolinium detection by syn-

chrotron XRF microscopy in human skin histological samples has

recently been applied in the study and diagnosis of a rare disease

associated with the administration of Gd-based CA in renal

patients.36 Other studies have also shown that these techniques are

useful to investigate Gd-based MRI and novel nanoparticles at single

cell and tissue level.37,38 Certainly, the ionizing nature of X-rays puts

some constraints on biomedical applications. The administered radia-

tion dose varies from 5 lGy for our low-resolution samples up to 106

Gy for the high-resolution samples. The latter is certainly too high

for in vivo applications; however, it is feasible for ex vivo studies. For

the quoted radiation doses, we have not observed any alteration of

the tissue as a result of the influence of the ionizing radiation, even

for the high-resolution mapping. In fact, repetitive element mapping

of the same sample delivered similar results (P � 0.01).

One drawback of synchrotron imaging, however, is the limited

access to the facilities, which does not allow large series of samples

to be analysed and longitudinal or repetitive measurements. How-

ever, the advantages of high sensitivity, chemical speciation and

mapping allow unique information to be obtained, thus facilitating

further investigations and speculations.

Overall, the present results show that SR-XRF elemental mapping

represents a new tool and a challenge in pharmacological and toxico-

logical investigations in animal models. We have shown that both

pharmacological distribution studies and diagnostic investigations

could greatly benefit from these X-ray spectroscopy techniques. The

possibility of investigating different length scales enables the moni-

toring of drug targeting from the organ to the cellular level, while

simultaneously correlating it with its physical and pathological status.

These techniques could thus open new perspectives and understand-

ing in hepatological and pharmacological research.

ACKNOWLEDGEMENTS

The authors acknowledge Marco Stebel for technical assistance in

animal studies. RD was recipient of a fellowship supported by a

MIUR grant (FIRB 2005-2009 RBIN04HC3S). We thank Kevin

Prince for careful reading of the manuscript.

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SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article: http://onlinelibrary.wiley.com/doi/10.1111/j.1440-

1681.2011.05618.x/suppinfo):

Figure S1. Layouts of the experimental set-up used at (a) the SYRMEP beam line at Elettra and at (b) the ID 21 beam line at the European Syn-

chrotron Radiation Facility (ESRF).

Figure S2. Calibration curve for Gd sensitivity recorded for the SYRMEP beam line at Elettra. The inlet depicts a typical spectra obtained for

the B2256 ⁄1 at a concentration of 100 lg ⁄mL of Gd.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any

queries (other than missing material) should be directed to the corresponding author for the article.




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