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Determination of urinary 8hplc-ed

Analytica Chimica Acta 516(2004)

107–110

Determination of urinary 8-hydroxy-2 -deoxyguanosine in obese

patients by HPLC with electrochemical detection

Eva Samcováa ,?,Petr Marhol a ,1,Franti?ek Opekar b ,Jan Langmaier c

3rd Faculty of Medicine,Centre of Biomedical Sciences,Charles University,Ruská87,10000Prague 10,Czech Republic b UNESCO Laboratory of Environmental Electrochemistry,Department of Analytical Chemistry,Charles University,

Albertov 2030,12840Prague 2,Czech Republic

c J.HeyrovskyInstitute of Physical Chemistry,Academy of Sciences of the Czech Republic,Dolej?kova 3,

18223Prague 8,Czech Republic

Received 6February 2004;received in revised form 19April 2004;accepted 19April 2004

Available online 2June 2004

Abstract

A simple amperometric detector with a platinum tubular electrode was tested for the determination of urinary 8-hydroxy-2 -deoxyguanosine (8-OHdG)by high-performance liquid chromatography.Analyte was extracted from human urine using a single-step solid-phase extraction with the average recovery of 71±7%.Analysis was carried out on a C-18analytical column under isocratic conditions.The limit of detection of 8-OHdG of 3nM (S /N =3)was comparable with that achieved using the conventional amperometric detector.Procedure was used for the determination of 8-OHdG levels in 10obese patients (body-mass index >30kg m ?2)yielding the mean concentration 32±27.1nM of 8-OHdG or 1.85±0.72?mol 8-OHdG mol ?1creatinine (4.63±1.8?g 8-OHdG g ?1creatinine),which is not much different from the levels found in healthy individuals.

Keywords:8-Hydroxy-2 -deoxyguanosine;DNA;Amperometric detector;Obese patient

1.Introduction

Free radical damage is an important factor in many pathological and toxicological processes [1].Considerable attention has been paid to 8-hydroxy-2 -deoxyguanosine (8-OHdG)as the biomarker of in vivo oxidative DNA dam-age [2,3].A sensitive determination of 8-OHdG in urine is possible using HPLC with the electrochemical detection (ED)[2–6].Various procedures have been developed for the puri?cation of the urine samples comprising the urine fractionisation by anion exchange chromatography [4],one-step [5,7,8],two-step [9]or multiple [2,3]solid-phase extraction,which was combined with the single [2,3,9],coupled [7,8]or triple [10]column HPLC–ED analysis.The electrochemical detection,which is based on the am-perometry of the electrochemical oxidation of 8-OHdG

Corresponding author.Tel.:+42-2-67102407;fax:+42-2-67102460.E-mail address:eva.samcova@lf3.cuni.cz (E.Samcov′a ).

1Present address:Department of Analytical Chemistry,Institute of Chemical Technology,Technick′a 5,16628Prague 6,Czech Republic.at a glassy carbon electrode,has been shown to be three orders of magnitude more sensitive than UV absorbance [11,12].Using HPLC–ED method,8-OHdG can be deter-mined in human urine with a detection limit of ca.1nM [2,3,8,9].

Recently,a novel amperometric detector for HPLC has been tested both in conventional and micro-HPLC systems.The detector is based on the tubular concept and differs from classical electrochemical detectors commonly used in liquid chromatography (thin-layer,wall-jet)in the design of the detection space.Since the detector contains no minia-turised components,its construction is very simple and easy to operate.An essential advantage of this detector is the ef-fective detection volume,which can be as low as ca.1nl [13].The aim of this work was to examine the suitability of the Pt tubular detector for the determination of the uri-nary 8-OHdG.We shall show that a sensitive determination can be based on a single-step extraction and HPLC analysis with a Pt tubular ED.The procedure will be used to deter-mine the urine levels of 8-OHdG in obese patients with an increased body-mass index.Obesity is a risk factor that has

108 E.Samcov′a et al./Analytica Chimica Acta 516(2004)107–110

an effect on the production of radical oxygen species and,

hence,of 8-OHdG [14].

2.Experimental 2.1.Chemicals

All chemicals were of analytical reagent grade quality or higher.Acetonitrile G Chromasolv for HPLC (Riedel–De Ha?n)and 8-OHdG were purchased from Sigma-Aldrich (Sigma-Aldrich Chemie GmbH,Germany).Ammonium ac-etate was purchased from Lachema Brno (Czech Republic).Aqueous stock solution of 35.3?M 8-OHdG as well as aque-ous solutions of 6%methanol and 10%acetonitrile were prepared using deionised water from DEMIW A 3water pu-ri?cation system (Watek,Czech Republic).Stock solution was stored at ?25?C.2.2.Urine clean-up procedure

Morning urine samples from healthy individuals or the obese patients were centrifuged at 1500×g at 10?C for 5min before processing to remove precipitates [9].The su-pernatant was then cleaned-up by a single-step solid-phase extraction.For the solid-phase extraction,Supelclean C-18SPE (6ml,500mg)columns were used.The columns were attached to a 12-port vacuum manifold (Supelco,USA).The sorbent of columns was preconditioned with 5ml of methanol and 5ml of water.Two millilitres of the super-natant with the aqueous 8-OHdG standard added was al-lowed to ?ow through the column under no excess vacuum.The column was then washed with 3ml of 6%methanol in deionised water,and subsequently with 6ml of deionised water.8-OHdG was eluted with 2ml of 10%acetonitrile in deionised https://www.wendangku.net/doc/9a7841161.html,anic solvent was evaporated by purg-ing nitrogen for 60min at 30?C.Finally,the volume of the sample was adjusted with water to 0.5ml and 50?l of the sample was injected to chromatographic column.

Urinary creatinine was determined before clean-up pro-cedure according to Jaffe’s picric acid method [15].2.3.Chromatographic analysis

A Shimadzu modular liquid chromatograph was used (Shimadzu Corporation,Kyoto,Japan),which was equipped with the autoinjector (SIL-10ADVp),the on-line degasser

(DGU-12A),the column oven (CTO-10AD),and the data station (CLASS VP 4.3).A Supelco Discovery C-18column (5?m particle size,250mm ×4.6mm i.d.)was used for LC separation of 8-OHdG.The mobile phase for isocratic elution of 8-OHdG was composed of 50mM ammonium acetate (pH 3.5)and 2%acetonitrile.

Flow rate was 0.4ml min ?1and the temperature of anal-ysis was 30?C.Injection sample volume was 50?l.ED was operated on 200nA range at the potential of the Pt electrode

Fig.1.Scheme of the amperometric detector with the platinum tubular electrode:(1)outlet from the HPLC column;(2)platinum tubular elec-trode;(3,4)auxiliary and reference electrodes;(5)potentiostat;(6)elec-trolyte solution;(7)waste.Zoom:(a)platinum tube;(b)PTFE tubes;(c)tin-soldered contact to the platinum tube;(d)shrinkable tube.

of 0.8V (versus Ag|AgCl|sat.KCl),which was controlled by the conventional three-electrode potentiostat.The whole procedure starting with the urine sample collection and end-ing with HPLC–ED analysis took approximately 2.5h.2.4.Electrochemical detector

The construction of the electrochemical detector is shown in Fig.1.The base of the detector is a 15mm long Pt tube (0.3mm o .d .×0.15mm i.d.,Chrompack)that is inserted into the PTFE tubing (1/16in.o .d .×0.010in.i.d.,Alltech).The PTFE tubing is connected to the output of the column using a standard ?tting.The other end of the Pt tube with PTFE overcoat,which serves as a working electrode of the three-electrode circuit,is dipped in a vessel containing the mobile phase electrolyte together with a Pt counter and an Ag|AgCl reference electrode.The detection space forms an integral part of the tubing outlet from the separation column,so that an additional peak broadening and sample zone di-lution are kept at minimum.A cyclic polarisation between the onsets of the oxygen and hydrogen evolution was used to activate the Pt electrode when necessary.Detector geom-etry and its working conditions were optimised for the de-termination of 8-OHdG.The optimisation consisted in the setting of suitable length of the shielding PTFE tube deter-mining the electroactive length of the Pt tubular electrode and in geometric arrangement of the auxiliary and reference electrodes in the detector cell.Depending on the cell geom-etry,detector operational parameters (working potential,the sequence and amplitude of activation pulses)were also var-ied to obtain the lowest S/N ratio.The optimum electrode potential was found by voltammetry of 8-OHdG on a Pt mi-croelectrode.V oltammetry was also used to check the lin-earity of the oxidation current with respect to the 8-OHdG concentration.The effective electrode area and the detec-tion volume of the tubular detector with the zero length of the PTFE shielding sleeve have been previously estimated as 6×10?3cm 2and 23.5nl,respectively [13].

E.Samcov′a et al./Analytica Chimica Acta 516(2004)107–110109

0.0

0.2

0.4

0.6

0.8

1.0

0.00

0.250.500.751.00I / n A

E / V

Fig.2.V oltammetric curve of 8-OHdG oxidation on Pt microelectrode at

various 8-OHdG concentrations in McIlvain buffer (pH 5):(···)0mM,(---)0.2mM,and (—)0.4mM;sweep rate:20mV s ?1.

2.5.Voltammetric measurements

V oltammetric measurements of the 8-OHdG oxidation on Pt microelectrode (10?m in diameter;BAS,USA)were carried out at the ambient temperature in an all-glass cell by using the three-electrode potentiostat (Model 273A,EG&G PAR),which was equipped with the operating soft-ware (M270,EG&G PAR).A Pt wire and the Ag|AgCl|sat.KCl electrode were used as the counter and the reference electrode,respectively.

3.Results and discussion 3.1.Voltammetry

A typical voltammogram of the 8-OHdG oxidation on Pt microelectrode is shown in Fig.2.On a microdisc,the voltammetric curve is expected to have the shape of a wave,with the limiting current I lim given by [19]I lim =4nFDr 0c 0

(1)

where n is the number of electrons consumed in the summary electrode reaction,D the diffusion coef?cient,c 0the bulk concentration of the electroactive species,and r 0the radius of the microdisc.In an agreement with Eq.(1),the limit-ing current is proportional to the concentration of 8-OHdG.Coulometric analysis in the potential range of the limiting current provided n =2±0.1.Based on Eq.(1),the slope of the concentration dependence of the limiting current,I lim (nA )=?0.04764+1829.58c (M ),corresponds to the diffusion coef?cient D =5×10?6cm 2s ?1.The half-wave potential E 1/2exhibits a linear dependence on the solution pH,E 1/2=0.77–0.023pH (in V).The electrochemical be-haviour of 8-OHdG on Pt is similar

to that observed on glassy carbon [16,17],and the mechanism of the electrode reaction is probably the same [18].

Fig.3.HPLC–ED chromatogram of urine sample of an obese patient containing 16nM 8-OHdG.

3.2.HPLC–ED analysis

HPLC–ED chromatogram of urine of an obese patient is shown in Fig.3.Quanti?cation of 8-OHdG in urine was based on the peak-high measurement using the linear re-gression curve for the aqueous standard solutions.Results showed to be linear over the range 50–500nM,with the re-gression equation y (a .u .)=(63.3±1.8)c (nM )?(62.4±331.8),the correlation coef?cient R =0.999and the stan-dard deviation of the ?t S .D .=337,which were evaluated with the help of a commercially available software (Origin 6.0,Microcal Software Inc.,USA).Under the conditions de-scribed,the retention time for 8-OHdG was 15.8±0.2min,which was checked by HPLC analysis of the urine sample after the SPE extraction,to which the standard solution of 8-OHdG was added.As compared with the sensitivity of the direct electrochemical determination of 8-OHdG on a Pt microelectrode (ca.1nA at 0.5mM),the sensitivity of HPLC–ED analysis (ca.1nA at 100nM)is approximately 5×103higher.Higher sensitivity is essentially due to the higher effective electrode area of the Pt tubular detector (6×10?3cm 2)compared to the area of the microelectrode (3×10?6cm 2),the diffusion layer thickness being approx-imately the same.This can be seen on comparing Eq.(1)with the corresponding equation for a tubular electrode [19]:

I lim =1.6nF

DA r 0 2/3v 1/3

c 0(2)

where A is the area of the tubular electrode with the radius r 0,and v is the volume ?ow rate.

Previous reports have indicated that the recovery of 8-OHdG in urine upon the solid-phase extraction is not as high as that in aqueous standards [20].In order to determine the recovery of 8-OHdG in urine,four test samples con-taining 8-OHdG (57.8,113.8,196.6and 320.9nM)were prepared from blank urine of healthy individuals by adding the standard solution of 8-OHdG before the solid-phase

110 E.Samcov′a et al./Analytica Chimica Acta516(2004)107–110 Table1

Precision and accuracy of the determination of8-OHdG in urine

Added c(8-OHdG)(nM)Measured c(8-OhdG)a(nM)CV(%)

57.859.6(6.9)11.6

113.8110.0(6.2) 5.6

196.6193.8(3.2) 1.6

320.9316.7(8.1) 2.6

a Mean(S.D.)(n=4).

extraction.The accuracy and precision of the extraction

procedure is given in Table1.The recovery of the target

analyte was found to be71±7%(n=16)and was found

to be constant over the concentration range58–320nM.In

contrast,the recovery of aqueous standards after the col-

umn extraction step was found to be86±5%(n=6).The

coef?cient of variation between series of measurement of

the100nM8-OHdG aqueous standard,stored for1week

at4?C and dark place,was7.5%.

The limit of detection(LOD),i.e.the concentration that

gives the signal equal to three times the noise level of de-

tector,was estimated as3nM.This value is comparable

with the detection limit reached previously using the con-

ventional amperometric detector[2,3,8,9].Mean8-OHdG

levels in random urinary samples from healthy individuals

were found to be,e.g.,14.6±7.5nM(129individuals)[8]

or29.2±21.6nM(60individuals)[9].Hence,the present

procedure appears to be suitable for examination of urine

samples with both normal and enhanced8-OHdG levels.

To test the procedure,we examined the urine samples col-

lected from both healthy individuals(?ve women and one

man,aged25–35years)and obese patients(10women aged

37–49years)with the body-mass index(BMI)higher than

30kg m?2.The body-mass index is de?ned as the body

weight(kg)divided by the body height squared(m2),and

the value over30kg m?2de?nes the obesity.The8-OHdG

level in urine of healthy individuals were found to be only

slightly above LOD=3nM,and could not be reliably

determined.The results of the determinations in urine of

obese patients are summarised in Table2.Mean concen-

tration of8-OHdG in these samples was32±27.1nM

Table2

Determination of8-OHdG in urine of10obese patients with the body-mass

index(BMI)exceeding30kg m?2

Patient no.Age

(years)

BMI

(kg m?2)

c(8-OHdG)

(nM)

c(8-OHdG)

(?mol mol?1creatinine)

13938.626.4 1.74 24543.911.70.96 33738.426.1 2.51 43943.932.7 2.44 53936.686.4 2.79 63745.310.10.96 74647.29.1 1.49 84954.515.7 1.06 94339.530.6 2.64 103742.875.7 1.90or1.85±0.72?mol8-OHdG mol?1creatinine,i.e.4.63±1.8?g8-OHdG g?1creatinine(1?mol8-OHdG mol?1cre-atinine=2.50?g8-OHdG g?1creatinine).Based on the comparison with the literature data[8,9],we conclude that the8-OHdG levels in urine of obese patients are not much different from those found in healthy individuals.

4.Conclusions

Simple one-step solid-phase extraction of the urine sample and the conventional HPLC with the Pt tubular detector can be reliably used for indirect measurements of oxidative DNA damage in humans with the8-OHdG urinary levels higher than3nM.Mean concentration of8-OHdG in urine samples collected from obese patients was not much different from the levels found in healthy individuals. Acknowledgements

This work was supported by the Grant Agency of the Czech Republic,grant No.203/01/0653,and partially by the Ministry of Education,Youth and Sports,Czech Republic, Research Project No.J13/98:111200001.We appreciate technical assistance by D.Bindzarová.

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关于人英语演讲稿(精选多篇) 关于人的优美句子 1、“黑皮小子”是我对在公交车上偶遇两次的一个男孩的称呼代号。一听这个外号，你也定会知道他极黑了。他的脸总是黑黑的；裸露在短袖外的胳膊也是黑黑的；就连两只有厚厚耳垂的耳朵也那么黑黑的，时不时像黑色的猎犬竖起来倾听着什么；黑黑的扁鼻子时不时地深呼吸着，像是在警觉地嗅着什么异样的味道。 2、我不知道，如何诠释我的母亲，因为母亲淡淡的生活中却常常跳动着不一样的间最无私、最伟大、最崇高的爱，莫过于母爱。无私，因为她的爱只有付出，无需回报；伟大，因为她的爱寓于

普通、平凡和简单之中；崇高，是因为她的爱是用生命化作乳汁，哺育着我，使我的生命得以延续，得以蓬勃，得以灿烂。 3、我的左撇子伙伴是用左手写字的，就像我们的右手一样挥洒自如。在日常生活中，曾见过用左手拿筷子的，也有像超级林丹用左手打羽毛球的，但很少碰见用左手写字的。中国汉字笔画笔顺是左起右收，适合用右手写字。但我的左撇子伙伴写字是右起左收的，像鸡爪一样迈出田字格，左看右看，上看下看，每一个字都很难看。平时考试时间终了，他总是做不完试卷。于是老师就跟家长商量，决定让他左改右写。经过老师引导，家长配合，他自己刻苦练字，考试能够提前完成了。现在他的字像他本人一样阳光、帅气。 4、老师，他们是辛勤的园丁，帮助着那些幼苗茁壮成长。他们不怕辛苦地给我们改厚厚一叠的作业，给我们上课。一步一步一点一点地给我们知识。虽然

他们有时也会批评一些人，但是他们的批评是对我们有帮助的，我们也要理解他们。那些学习差的同学，老师会逐一地耐心教导，使他们的学习突飞猛进。使他们的耐心教导培养出了一批批优秀的人才。他们不怕辛苦、不怕劳累地教育着我们这些幼苗，难道不是美吗？ 5、我有一个表妹，还不到十岁，她那圆圆的小脸蛋儿，粉白中透着粉红，她的头发很浓密，而且好像马鬓毛一样的粗硬，但还是保留着孩子一样的蓬乱的美，卷曲的环绕着她那小小的耳朵。说起她，她可是一个古灵精怪的小女孩。 6、黑皮小子是一个善良的人，他要跟所有见过的人成为最好的朋友！这样人人都是他的好朋友，那么人人都是好友一样坦诚、关爱相交，这样人与人自然会和谐起来，少了许多争执了。 7、有人说，老师是土壤，把知识化作养分，传授给祖国的花朵，让他们茁壮成长。亦有人说，老师是一座知识的桥梁，把我们带进奇妙的科学世界，让