Plasma phospholipid metabolic profiling and biomarkers of rats following radiation exposure based on liquid chromatography–mass spectrometry technique

Research Article

Received: 5 December 2008, Accepted: 26 January 2009 Published online in Wiley Interscience: 21 April 2009

(www.interscience.wiley.com) DOI 10.1002/bmc.1226

Biomed. Chromatogr. 2009; 23: 1079–1085 Copyright © 2009 John Wiley & Sons, Ltd.

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John Wiley & Sons, Ltd.

Plasma phospholipid metabolic profiling and biomarkers of rats following radiation exposure based on liquid chromatography–mass spectrometry techniquePhospholipid metabolic profiles and biomarkers

Chang Wang,a* Jun Yangb and Jihua Niea

ABSTRACT: Lipidomics, a prominent area of metabolomics, utilizes novel analytical methodologies to study the extensive classesof lipid molecules, changes in lipid metabolism and lipid-mediated signaling processes. In this paper, the phospholipid metabolicprofiles changes and potential biomarker identification in the rats plasma after γ-irradiation exposure were investigated bycoupling high performance liquid chromatography–mass spectrometry technology to multivariate statistical analysis. Orthogonalpartial least-squares to latent structures discriminate analysis (OPLS-DA) was used to find the potential plasma phospholipidsbiomarkers of rats for radiation exposure. According to the corresponding tandem mass spectrometric results, potential biomarkerswere identified. After exposure to γγγγ-rays, phosphatidylethanolamine and phosphatidylserine showed a marked increase, andphosphatidylcholine, sphingomyelin and lysophosphatidylcholine followed the same trend, although their rise was not sig-nificant. The results suggested that radiated rats had a phospholipid metabolic abnormality, which could be an alternativeway to assess the radiaton exposure. The biomarkers may involve a radiation-induced apoptosis pathway and represent apromising target for discovery new radioprotective drugs and radiosensitizers. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: phospholipid metabolic profiles; biomarkers; radiation exposure

Introduction

Radiation exposure has both short- and long-term adverse health

effects. People may be exposed to ionizing radiation during

radiotherapy or following exposure to radionuclides in nuclear

medicine. Therefore, it is necessary to develop biomarkers of

ionizing radiation exposure that can be used as target for radio-

protective agents which can reduce morbidity or mortality pro-

duced by ionizing irradiation. The search for biomarkers of the

early effects of ionizing radiation exposure in both humans and

experimental animals has a history spanning several decades.

Blood cells, serum and urine have proven to be rich sources of

human radiation biomarkers, including those of DNA damage

and repair (Garaj-Vrhovac and Kopjar, 2003; Plappert et al., 1997),

chromosomal aberrations (Blakely et al., 2001), DNA–protein

cross links (Budhwar et al., 2003), red blood cell polyamine levels

(Porciani et al., 2001), serum proteomic profiles (Menard et al.,2006), gene expression profiles determined by both microarrays

(Amundson et al., 2004), RT-PCR (Kang et al., 2003), N-hexanoylglycine

and taurine excretion in urine (Tyburski et al., 2008). Several bio-

logical parameters of animal plasma including cholesterol, phos-

pholipids and so on have been extensively investigated. The

changes in cholesterol, phospholipids and triglyceride fractions

in the plasma of rabbits and rats after irradiation had been

published (Feurgard et al., 1998; Di Luzio and Simon, 1957; Rizvi

et al., 1984; Feliste et al., 1981; Yousri et al., 1991). However, studies

on plasma phospholipid species profiling alterations and biomar-

kers of irradiated rats have not been investigated.

Phospholipids are the main components of membranes and

perform important biological functions (Marcus and Hajjar,

1993; Berridge, 1993). Many metabolites derived from their deg-

radation are important intracellular signaling molecules involved in

processes such as proliferation and apoptosis. Additionally, most

phospholipid classes have different polar head groups and each

class contains different combinations of fatty acids in the sn-1and sn-2 position of the glycerol backbone. Owing to phospho-

lipid structural diversity, liquid chromatography/electrospray

ionization mass spectrometry (LC/ESI-MS) has been a very useful

tool for phospholipid analysis (Kim and Salem, 1993; Delong

et al., 2001; Wang et al., 2005; Gao et al., 2006). The composition

and level of phospholipids in biological are altered by physio-

logic and pathologic states (He et al., 2007; Stein and Stein, 2005;

Walter et al., 2004).

* Correspondence to: Chang Wang, Jiangsu Provincial Key Laboratory of Radi-

ation Medicine and Protection, School of Radiation Medicine and Public

Health, Medical College of Soochow University, Suzhou Industrial Park

Ren’ai Road 199, Suzhou 215123, People’s Republic of China. E-mail:

[email protected]

a Jiangsu Provincial Key Laboratory of Radiation Medicine and Protection,

School of Radiation Medicine and Public Health, Medical College of Soo-

chow University, Suzhou 215123, People’s Republic of China

b University of California, Department of Entomology, Cancer Research

Center, One Shields Avenue, Davis, CA 95616, US

Abbreviations used: lysoPC, lysophosphatidylcholine; OPLS, orthogonal

PLS; OPLS-DA, OPLS discriminate analysis; PC, phosphatidylcholine; PCA,

principal components analysis; PE, phosphatidylethanolamine; PLS, partial

least-squares to latent structures; PS, phosphatidylserine; SM, sphingomyelin.

Contract/grant sponsor: Medical Development Fund of Soochow University;

Contract/grant number: EE126714

C. Wang et al.

www.interscience.wiley.com/journal/bmc Copyright © 2009 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2009; 23: 1079–1085

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Metabonomics has been defined as ‘the quantitative mea-

surement of the dynamic multiparametric response of a living

system to pathophysiological stimuli or genetic modification’

(Nicholson et al., 1999, 2002). Recently, LC/MS has become a

major tool for metabonomics research (Theodoridis et al., 2008;

Lu et al., 2008). Lipidomics, a prominent area of metabolomics,

utilizes novel analytical methodologies to study the extensive

classes of lipid molecules, changes in lipid metabolism and lipid-

mediated signaling processes (Wolf and Quinn, 2008). Lipidomics,

integrated with genomics, proteomics and metabolomics, con-

tributes to understanding how lipids function in a biological system

and provides a powerful tool for elucidating the mechanisms of

lipid-based diseases, for biomarker screening and for monitoring

pharmacologic therapy (Watson, 2006). In order to summarize

and interpret the complex data, efficient and robust modeling,

analysis and interpretation methods are needed. The popular

ones included principal components analysis (PCA) (Jackson, 1991),

partial least-squares to latent structures (PLS) (Wold et al., 2001)

and orthogonal PLS (OPLS) (Trygg and Wold, 2002) . Recently, an

OPLS discriminate analysis (OPLS-DA) model has been success-

fully applied to identify biochemically interesting compounds

(Wiklund et al., 2008; Bylesjo et al., 2006).

In this work, LC/MS technology combined with multivariate

statistical analysis was employed to monitor the rats plasma

phospholipid metabolic profiles changes after γ-irradiation expo-

soure. Potential biomarkers were discovered and identified by

the tandem mass spectra. The result not only suggests that radi-

ation exposure can cause phospholipid metabolic disorder, but

also indicates that plasma phospholipids profiles could be a

promising way to assess the radiation exposure. The biomarkers

identified here are related to multiple biological processes

including the cell apoptosis pathway. In addition, the biomarkers

here could be potentially used as the target for radioprotective

drugs and radiosensitizers.

Material and Methods

Regents

1,2-Dipentadecanoyl-sn-glycero-3-phosphoethanolamine (C15:0/

C15:0 PE), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium

salt) (C14:0/C14:0 PS, phosphatidylserine), 1,2-dipentadecanoyl-

sn-glycero-3-phosphocholine (C15:0/C15:0 PC) and 1-heptadecanoyl-

2-hydroxy-sn-glycero-3-phosphocholine (C17:0 lysoPC) were from

Avanti Polar Lipids (Alabaster, AL, USA) and were used as internal

standards. Other phospholipid standards were from Avanti Polar

Lipids or Sigma (St Louis, MO, USA). 2,6-Di-tert-butyl-4-methylphenol

was from Aldrich-Chemie (Steinheim, Germany). Formic acid and all

the solvents were HPLC grade (Tedia, USA); ammonia was analytical

grade from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China).

Animals and Irradiations

Male Sprague–Dawley rats (from Soochow University Laboratory

Animal Center, China) were randomized in two groups: control

and irradiated (each group contains eight animals). Rats were

exposed to 60Co γ-rays at a dose rate of 1 Gy/min with a distance

of 3 m from the radioactive source. After 24 h of irradiation, blood

was collected from the heart under chloral hydrate. The plasma

samples were obtained by centrifugation at 2200g for 15 min at

4°C, and then kept at −80°C until analysis. All experiments were

conducted according to the Chinese regulations for animal

experimentation (Commission on Science and Technology, State

Bureau of Technical Supervision, no. 593, 11 December 1997).

Sample Preparation

The phospholipid standards were dissolved (approx. 1 mg/mL)

in chloroform–methanol (2:1, v/v), and further diluted in hexane–

1-propanol (3:2, v/v). The lipids in the plasma samples were extracted

according to modified Folch method. In brief, 8 mL chloroform-

methanol (2:1, v/v) containing 0.01% 2,6-di-tert-butyl-4-

methylphenol and appropriate amounts of internal standards

(e.g. C15:0/C15:0 PE, C14:0/C14:0PS, C15:0/C15:0 PC, C17:0 lysoPC)

were added to 300 μL plasma, then the solution was vortex-mixed

for 1 min and incubated for approximately 0.5 h at room tempera-

ture. Finally, 1.3 mL KCl (50 mmol/L) was added, mixed and centri-

fuged at 2000g for 15 min, then the upper phase was washed

with 2 mL chloroform, and the two organic phases were combined

and dried under nitrogen. The samples were stored at −20°C.

Before analysis, the extracted samples were reconstituted in 300 μL

of chloroform–methanol (2:1, v/v) solution and then diluted 10

times with hexane–1-propanol (3:2, v/v).

High-performance liquid chromatography and mass

spectrometry

An HP 1100 series HPLC system (Agilent Technologies, Palo Alto, CA,

USA) was used. The HPLC separation was performed on a diol

column (Nucleosil, 100-5 OH, Germany), 250 (mm) × 3.0 (mm, i.d.) ×5.0 (μm, particle size). The total flow rate was 0.4 mL/min. The

column temperature was kept at 35°C. The linear solvent gradi-

ent started at 32% B and 68% A, at 20 min was linearly increased

to 80% B, then held for 13 min, over 5 min linearly decreased to

32% and finally held for another 22 min (Wang et al., 2004). Solvent

mixture A was hexane–1-propanol–formic acid–ammonia (79/

20/0.6/0.07, v/v); solvent mixture B was 1-propanol–water–formic

acid–ammonia (88/10/0.6/0.07, v/v).

The mass spectrometric detection was conducted on a QTRAP

LC/MS/MS system from Applied Biosystems/MDS Sciex (USA)

equipped with a turbo ionspray source. The combination of

highly selective triple-quadrupole MS-MS scans and high-sensitivity

ion trap product scans on the same instrument platform pro-

vided rapid identification of phospholipids of extracted rat

plasma sample. The detection of phospholipids eluted from the

chromatographic column was performed in the ‘enhanced MS’

(EMS), single quadrupole mode (so called ‘survey scan’) where

ions were accumulated and then filtered in the Q3-linear ion-

trap. The structure of the phospholipid was elucidated by the

‘enhanced’ product ion scan mode (EPI), where ions were

trapped in the third quadrupole before filtration.

Data Analysis

Negative-ion LC/MS chromatograms were inspected for profiling

the phospholipid species in plasma. Masses corresponding to

the quasi-molecular anions [M − H]− [for phosphatidylinositol (PI),

phosphatidylethanolamine (PE) and phosphatidylserine (PS)] or

[M − 15]− [for phosphatidylcholine (PC), sphingomyelin (SM) and

lysophosphatidylcholine (lysoPC) species] for each phospholipid

species were plotted against elution time. From the LC/MS profile

of a plasma sample, phospholipid species constantly occurring

in all rat plasma samples were collected to form a data array. The

quantification of a class of lipid molecular species in lipid extracts

Phospholipid metabolic profiles and biomarkers

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can be made by comparisons of the individual molecular ion

peak intensity with internal standard after correction for 13C iso-

tope effects (Han and Gross, 2001). For PI and SM species, due to

the lack of commercial internal standards, their quasi-quantifications

were based on PE and PC internal standards, respectively. OPLS-

DA was used to find potential biomarkers for radiation exposure.

Multivariate analysis was performed using the SIMCA-P 11 demo

version (Umetrics AB, Umeå, Sweden). In addition, to improved

performance of multivariate pattern-recognition analysis and

enhanced predictive power of the model, Pareto scaling was used

(Wold et al., 1993). Pareto scaling weighs each variable by the

square root of its standard deviation, which amplifies the con-

tribution of lower concentration metabolites but not to such an

extent that noise produces a large contribution. An S-plot was

used to look for the potential plasma phospholipid biomarkers

of rats for radiation exposure.

Results and Discussion

The full scan of phospholipid species in rat plasma was in the

negative-ion mode because most phospholipid species in this

mode have relatively high sensitivity (Wang et al., 2004) with

less interference. Figure 1 illustrates the total ion current (TIC)

chromatography of rat plasma. PE was first eluted, followed by

PI/PS, PC, SM and lysoPC in a successive manner for phospholip-

ids containing a given fatty acyl composition. Because different

molecular species within a class have the same polar heads,

their retention times in HPLC are very close. The retention time

difference of compounds with one class the same is less than

that with two different classes, which can be used to align the

retention times of phospholipid species in the extraction ion

chromatography to avoid the retention time fluctuations

between different injections. From the TIC chromatography, PC

is the most abundant phospholipid class in the rat plasma, with

lysoPC being the next most prominent phospholipid, followed

by SM, PE, PI and PS.

After 24 h of whole-body irradiation of rats with doses of 3.5 Gy,

the changes of phospholipids level in the plasma were investigated

(Table1). There was a remarkable increase in the total phospho-

lipids level of plasma when rats were exposed to 3.5Gy dose

radiation, which was consistent with previous observations (Di

Luzio and Simon, 1957; Rizvi et al., 1984; Steadman and Thompson,

1950; Entenman et al., 1955). This indicated that ionizing radia-

tion could lead to phospholipid metabolism disorder. Among

the phospholipids, the PE level in the plasma increased almost

2.5 times and PS almost doubled, as compared with control rats

(Table 1). PC, SM and lysoPC followed the same trend, although

their increase was not significant.

Normalized by the corresponding internal standard, the data

was fed into SIMCA-P software for OPLS-DA analysis. The model

could describe 74.7% of the variation in x (r2x = 74.7%), 89.0% of

the variation in the response y (class) (r2y = 89.0%) and predict

79.9% of the variation in the response y (q2y = 79.9%). The satis-

fied classification ability between the irradiated rats and the

healthy controls can also be seen from Fig. 2(a), which also

Figure 1. Total ion chromatogram of LC/ESI-MS analysis of phospholipids of extracted rat plasma sample. Condi-

tions are given in the Experimental section.

Table 1. Effect of 3.5 Gy γ-irradiation on phospholipids

composition of plasma of Sprague–Dawley rats

Phospholipid Control Irradiated

Total phospholipids 31.91 ± 5.77 48.46 ± 11.90*

PE 8.29 ± 2.65 20.64 ± 6.18*

PI 4.36 ± 0.59 4.19 ± 1.00

PS 0.99 ± 0.45 1.82 ± 0.58†

PC 5.80 ± 0.77 6.06 ± 1.32

SM 7.30 ± 2.54 10.39 ± 3.51

lysoPC 5.16 ± 1.12 5.55 ± 0.73

Note: results are means ± SD, n = 8.†Significantly difference at p < 0.05 compared with control.

*Significantly difference at p < 0.001 compared with control.

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Figure 2. Related plots from OPLS-DA models classifying irradiated rats and healthy control. (a) Scores plot; the solid ellipse denotes the 95% signif-

icance limit. (�) Healthy control; (�) irradiated rats. (b) S-plot; the potential phospholipids biomarkers are highlighted by red squares. (c) Loading plot

with jack-knifed confidence intervals. (d) VIP plot.



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indicates that phospholipids metabolism disorder occurred in

irradiated rats. S-plot combines the contribution/covariance

[Cov(t,x)] and reliability/correlation [Corr(t,x)] from OPLS-DA

model and is a good method for biomarker discovery (Wiklund

et al., 2008). Statistically significant phospholipid species account-

ing for differentiating between control healthy and radiated rats

were selected from the S-plot [Fig. 2(b)] together with the jack-

knifed confidence interval (CIJFJK) [Fig. 2(c)] and VIP (Variable

Importance in the Projection) plot (Fig. 2d). The loading plot with

CIJFJK displayed the uncertainty of each variable and the smaller

span of confidence interval gives more creditability to the selected

variable. Then, those variables with CIJFJK across zero were excluded.

In this study, multi-criteria assessment (Ni et al., 2008) for multi-

variate statistics was used to identify phospholipids biomarkers

between the irradiated rats and healthy controls. Then, the vari-

ables meeting all three criteria [i.e. VIP > 1, |Corr(t,x)| > 0.60, and

the span of CIJFJK excluding zero] were selected as potential

biomarkers for radiation injury.

The identification of these biomarkers was carried out by EPI

experiment under the negative mode. As an example, Fig. 3(a)

shows the negative EPI spectra of the compound with m/z 750.6

in the negative-ion mode. Its retention time shows that it belongs

to PE species. The presence of a relatively more intense lysoplas-

menylethanolamine-like ion peak (m/z 464.5, C18:0 lysopPE; m/z

436.4, C16:0 lysopPE) indicates that it belongs to plasmalogen

PE (Wang et al., 2004). The fragment ions detected at m/z 303.3

and 331.3 corresponded to C20:4 and C22:4 fatty acid residues

(carboxylate anion fragments), respectively. The position of the

acyl chains in the glycerol backbone of the phospholipid molecule

is obviously important for their degree of dissociation. There has

been a discrepancy about which carboxylate anion yields the

most intense peak in the product ion spectrum (Wang et al.,2004). The phospholipids isolated from animals most often con-

tain a saturated fatty acid in sn-1 position and an unsaturated

fatty acid in sn-2 position (Yorek, 1993). Therefore, the [M − H]−

at m/z 750.6 was identified as pC18:0/C20:4 PE or pC16:0/C22:4

plasmalogen PE species. The possible structures are given in

Fig. 3(b).

Most of the biomarkers belong to PE; others are PS and SM

(Table 2). Compared with normal rats, all the biomarkers show a

remarkable increase in plasma of irradiated rats. At present,

the cause of the increase of phospholipids in plasma induced by

radiation injury is still not clear. As the main component part of

membranes, the important role of phospholipids in radiation-

induced apoptosis is the mostly likely reason. Radiation-induced

DNA damage can induce death by apoptosis by activation of

signal transduction pathways, such as the sphingomyelin/

ceramide signal transduction pathway (Vit and Rosselli, 2003;

Figure 3. (a) EPI spectrum of m/z 750.6 representing the [M − H]− ion of plasmalogen PE. (b) Structure of this compound.

C. Wang et al.


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Billis et al., 1998). Moreover, radiation can also led to the cell

surface exposure of PS and PE (Emoto et al., 1997; Marconescu

and Thorpe, 2008). Recently, the importance of PS and SM in cell

apoptosis and cell recognition has been emphasized (Hanshaw

and Smith, 2005; Green, 2000; Vance and Steenbergen, 2005; Fadok

et al., 2001). The exposure of PS on the outside surface of cells is

widely believed to play a key role in the removal of apoptotic

cells and in initiation of the blood clotting cascade. Furthermore,

PS is required as a cofactor for several important enzymes, such

as protein kinase C and Raf-1 kinase, which are both involved in

signaling pathways (Vance and Steenbergen, 2005). Using genetic

models of acid sphingomyelinase deficiency, the ceramide

generated by radiation-induced activation of sphingomyelinase

has been shown to serve as a second messenger in initiating an

apoptotic response (Billis et al., 1998). However, the significance

of PE in cell apoptosis remains to receive wide attention. The

possible mechanism of PE in apoptosis may relate to interrelated

metabolism between PE and PS. PE is made in mammalian

cells by two completely independent major pathways. In one

pathway, PS is converted into PE by the mitochondrial enzyme

PS decarboxylase (Borkenhagen et al., 1961). In another, PE is

made via the CDP-ethanolamine pathway (Tijburg et al., 1989).

In addition, coincident exposure of PE and PS on the surface of

irradiated cells also suggests that PE and PS share common

regulatory mechanisms of translocation (Marconescu and Thorpe,

2008).

Conclusion

In this work, phospholipids profiling analysis in plasma of rats

based on LC-MS was investigated to study ionizing radiation

injury. After exposure to γ-rays, PE and PS showed a marked

increase; PC, SM and lysoPC followed the same trend, although

their increase was not significant. These results suggest that

ionizing radiation can lead to phospholipid metabolism disorder,

and the plasma phospholipids profiles could be a promising way

to assess the radiation exposure. By coupling OPLS-DA multivar-

iate statistical analysis, some potential phospholipid biomar-

kers for radiosensitivity were discovered. The biomarkers may

involve radiation-induced apoptosis pathway and represent a

promising target for the discovery of new radioprotective drugs

and radiosensitizers.

Acknowledgements

This study was supported by the Medical Development Fund of

Soochow University (EE126714).

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Table 2. Potential phospholipids biomarkers as identified

in plasma of irradiated rats after exposing ionizing radiation

Ion Mass (m/z) Molecular species composition

[M − H]− 714.5 C16:0/C18:2 PE

[M − H]− 722.6 pC16:0/C20:4 PE

[M − H]− 738.6 C16:0/C20:4 PE

[M − H]− 742.6 C18:0–C18:2 PE

[M − H]− 746.5 C18:0–C18:0 PE

[M − H]− 748.5 pC16:0/C22:5 PE, pC18:1/C20:4 PE

[M − H]− 750.6 pC18:0/C20:4 PE, pC16:0/C22:4 PE

[M − H]− 762.5 C16:0/C22:6 PE

[M − H]− 764.6 C16:0/C22:5 PE, C18:1/C20:4 PE

[M − H]− 766.6 C18:0/C20:4 PE, C16:0/C22:4 PE

[M − H]− 774.6 pC18:0/C22:6 PE

[M − H]− 776.6 pC18:0/C22:5 PE

[M − H]− 778.6 pC20:0/C20:4 PE, pC18:0/C22:4 PE

[M − H]− 790.6 C18:0/C22:6 PE, C20:2/C20:4 PE

[M − H]− 834.6 C18:0/C22:6 PS

[M − CH3]− 685.5 34:2 SM

Note: pPE, plasmalogen PE.



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Plasma phospholipid metabolic profiling and biomarkers of rats following radiation exposure based on liquid chromatography–mass spectrometry technique