Measurement of urinary F2-isoprostanes by liquid chromatography-mass spectrometry method : method evaluation and application in health sciences

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Measurement of urinary F2-isoprostanes by liquid chromatography-mass spectrometry method : method evaluation and application in health sciences

 

Author: Ng, Tsz-kin Jacky
Title: Measurement of urinary F2-isoprostanes by liquid chromatography-mass spectrometry method : method evaluation and application in health sciences
Degree: M.Sc.
Year: 2009
Subject: Hong Kong Polytechnic University -- Dissertations.
Urine -- Analysis.
Mass spectrometry.
Liquid chromatography.
Department: Dept. of Health Technology and Informatics
Pages: ix, 135 leaves : ill. ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2303577
URI: http://theses.lib.polyu.edu.hk/handle/200/4542
Abstract: F2-isoprostanes are prostaglandin-like compounds that are produced in vivo independently of cyclooxygenase enzyme, primarily by free radical-induced peroxidation of arachidonic acid. They can be measured in a range of tissues and biological fluids including plasma, urine, cerebral spinal fluid, and exhaled condensate. F2-isoprostanes has been suggested to be a reliable indicator of lipid peroxidation that may be related to in vivo free radical generation, oxidative stress or damage and antioxidant deficiency. It is important as oxidative stress has been implicated in a wide variety of acute and chronic disease processes. In this study, the following work was performed to 1) set up and evaluate a novel method of measuring urinary F2-isoprostanes by using high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS); 2) investigate the concentration and biological variation of urinary F2-isoprostanes in a group of apparently healthy Chinese subjects; 3) apply the test method in health sciences by investigating the effect of green tea intake on urinary F2-isoprostanes concentration. Results: A simple, fast, sensitive and reliable HPLC-MS/MS urinary F2-isoprostanes assay was setup. Direct injection of 30uL of urine into HPLC-MS/MS system for analysis was performed, with no sample cleanup procedure required. The total analysis time per sample (start from sample injection until result obtained) is 25 minutes. The method proved to be linear up to at least 10000 ng/L (r2=0.9996). The absolute limit of detection (LOD) and the LOD in urine matrix were 20.2 ng/L and 28.6 ng/L respectively. The within-run and between-run CVs were <4.7% (n=6 at each of 3 different F2-isoprostane concentrations (500, 2500 and 5000 ng/L) and <5.4% (n=6 at each of 3 different F2-isoprostanes concentrations (500, 2500 and 5000 ng/L), respectively. The detection reproducibility (CV) of urinary F2-isoprostanes at room temperature over 20 hours was 4.1 % (n=6). The mean recovery of the F2-isoprostanes HPLC-MS/MS assay was 97.8% -104.5% (n=6) across five different F2-isoprostanes concentrations (625, 1250, 2500, 5000 and 10000 ng/L). The range of basal urinary F2-isoprostanes in 50 apparently healthy Chinese subjects was from 546.1 to 7520.3 ng/L(or from 133.1 to 481.1 pg/mg creatinine). The median, mean (SD) was 2757.5, 2977.5 (1482.4) ng/L (or 291.4, 304.3 (97.2) pg/mg creatinine). In age- and body mass index (BMI)-matched apparently healthy Chinese subjects, although urinary F2-isoprostanes (expressed as mean (SD) in pg/mg creatinine) in males (290.2 (92.5), n=25) was slightly lower than that of females (318.3 (101.7), n=25) but they showed no significant difference (p=0.3126). No significant correlation of urinary F2-isoprostanes with age (r2=0.057; p=0.0938) or with BMI (r2=0.019; p=0.3364) was detected. In addition, while within-individual variation was wide, no significant overall changes in F2-isoprostanes excretion within a single day (p=0.4193) or over five consecutive days (p=0.8861) were seen. The total analytical variation of determining creatinine-normalized urinary F2-isoprostanes with the current method was 5.8%. The overall biological between-individual and within-individual CVs of 23 apparently healthy Chinese individuals (11 males and 12 females) were 29.2% and 20.8% respectively. The index of individuality was 0.69 and the reference change value (RCV) was 58.3%. The within-individual biological variations were not related to the sex of the individuals (p=0.7796). Stratification according to gender did not significantly decrease the index of individuality or increase the utility of conventional population-based reference. For green tea intervention study, both brand A, Loongjin green tea (p=0.7662) and C, water control (p=0.6852) showed no significant difference in urinary F2-IsoPs before and after the intervention. Only the intervention B, Screw-shaped Green Tea seems to be able to lower the urinary F2-IsoPs level after the intake, however statistically it showed no significant difference (p=0.0517) before and after intervention. Difference of each intervention showed no significant difference (p= 0.1237) against the other intervention. Further investigation may be needed to establish a good relationship between Screw-shaped Green tea and F2-IsoP excretion. In conclusion, a specific, sensitive, reproducible, simple, automatic and fast HPLC-MS/MS urinary F2-isoprostanes assay was developed that met the desirable analytical performance goals, linearity, LOD, recovery and precision. The method is suitable for large scale clinical and epidemiological studies. We have defined preliminary population reference intervals for Hong Kong Chinese using the method, and these will support further studies using this novel biomarker of oxidative stress. Urinary F2-IsoPs HPLC-MS/MS assay can be applied to investigate the effect of antioxidant treatment in human subjects.

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