Urinary 8-isoPGF Research Articles

Determination of ω-6 and ω-3 PUFA metabolites in human urine samples using UPLC/MS/MS.

The ω-6 and ω-3 polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are the precursors of various bioactive lipid mediators including prostaglandins, thromboxanes, leukotrienes, hydroxyeicosatetraenoic acid, isoprostanes, lipoxins, and resolvins (Rvs). These lipid mediators play important roles in various physiological and pathological processes. The quantitative determination of PUFA metabolites seems necessary for disease research and for developing biomarkers. However, there is a paucity of analytical methods for the quantification of ω-6 and ω-3 PUFA metabolites—the specialized pro-resolving mediators (SPMs) present in the human urine. We developed a method for the quantification of ω-6 and ω-3 PUFA metabolites present in human urine using ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS). The developed method shows good linearity, with a correlation coefficient >0.99 for all of the analytes. The validation results indicate that our method is adequately reliable, accurate, and precise. The method was successfully used to examine urine samples obtained from 43 healthy volunteers. We could identify 20 PUFA metabolites, and this is the first report of the quantitative determination of RvD1, 17(R)-RvD1, 11-dehydro thromboxane B3, RvE2, and 5(S)-HETE in human urine. The urinary 8-iso PGF(2α) and PGE2 levels were significantly higher in the men smokers than in the men nonsmokers (p < 0.05). In this study, we developed an accurate, precise, and novel analytical method for estimating the ω-6 and ω-3 PUFA metabolites, and this is the first report that the SPMs derived from EPA and DHA are present in human urine.

Oxidative stress early in infancy and neurodevelopmental outcome in very low‐birthweight infants

Reactive oxygen species may be involved in serious diseases in premature infants. The objective of this study was to assess the relationship between neurodevelopmental outcome and oxidative stress marker level in the urine of very low-birthweight (VLBW) infants. Spot urine samples were collected from 35 VLBW infants. Urinary excretion of 8-hydroxy-2″-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, and 8-iso-prostaglandin F2α (8-isoPGF), a marker of lipid peroxidation, was measured at 1, 2, 4, and 6 weeks of age. Neurodevelopmental outcome at 18 months' corrected age was assessed using the Bayley Scales of Infant Development (BSID)-II. Significant correlations were found between urinary 8-OHdG at 2 and 4 weeks and the Mental Development Index of the BSID-II. No significant correlation was found between urinary 8-isoPGF and indices of the BSID-II. In VLBW infants, urinary 8-OHdG level correlated with mental development rather than psychomotor development at 18 months' corrected age; urinary 8-OHdG might be a predictive marker of neurodevelopmental outcome in VLBW infants.

Quantitative determination of 8-isoprostaglandin F 2α in human urine using microfluidic chip-based nano-liquid chromatography with on-chip sample enrichment and tandem mass spectrometry

Urinary 8-isoprostaglandin F 2α (8-isoPGF 2α) has been reported as an important biomarker to indicate the oxidative stress status in vivo. In order to quantitatively determine the low contents of 8-isoPGF 2α (in sub- to low ng mL −1 range) in physiological fluids, a sensitive detection method has become an important issue. In this study, we employed a microfluidic chip-based nano liquid chromatography (chip-nanoLC) with on-chip sample enrichment coupled to triple quadrupole mass spectrometer (QqQ-MS) for the quantitative determination of 8-isoPGF 2α in human urine. This chip-nanoLC unit integrates a microfluidic switch, a chip column design having a pre-column (enrichment column) for sample enrichment prior to an analytical column for separation, as well as a nanospray emitter on a single polyimide chip. The introduction of enrichment column offers the advantages of online sample pre-concentration and reducing matrix influence on MS detection to improve sensitivity. In this study, the chip-nanoLC consisting of Zorbax 300A SB-C18 columns and Agilent QqQ Mass spectrometer were used for determining 8-isoPGF 2α in human urine. Gradient elution was employed for effective LC separation and multiple reaction monitoring (MRM) was utilized for the quantitative determination of 8-isoPGF 2α ( m/ z 353 → 193). We employed liquid–liquid extraction (LLE)/solid-phase extraction (SPE) for extracting analyte and reducing matrix effect from urine sample prior to chip-nanoLC/QqQ-MS analysis for determining urinary 8-isoPGF 2α. Good recoveries were found to be in the range of 83.0–85.3%. The linear range was 0.01–2 ng mL −1 for urinary 8-isoPGF 2α. In addition, the proposed method showed good precision and accuracy for 8-isoPGF 2α spiked synthetic urine samples. Intra-day and inter-day precisions were 1.8–5.0% and 4.3–5.8%, respectively. The method accuracy for intra-day and inter-day assays ranged from 99.3 to 99.9% and 99.4 to 99.7%, respectively. Due to its rapidity, enhanced sensitivity, and high recovery, this chip-nanoLC/QqQ-MS system was successfully utilized to determine the physiological biomarkers such as 8-isoPGF 2α in human urine for clinical diagnosis.

Oxidative stress in midpregnancy as a predictor of gestational hypertension and pre‐eclampsia

To explore the relationship between the levels of maternal oxidative stress and glycaemia during pregnancy and to compare the predictive values of 8-epimer of prostaglandin F(2alpha) (8-isoPGF(2alpha)) and mean arterial pressure (MAP) in midpregnancy for the development of hypertensive complications in later pregnancy. Prospective observational study as an ancillary study to the Hyperglycaemia and Adverse Pregnancy Outcome (HAPO) study. Obstetric clinics and wards of a university teaching hospital in Hong Kong. Selected women with singleton pregnancies attending the antenatal clinic. Pregnant women who met HAPO inclusion criteria were recruited for the study. Glucose tolerance was assessed by a 75-g 2-hour oral glucose tolerance test (OGTT) at 24-32 weeks of gestation. Fasting plasma samples for 8-isoPGF(2alpha) estimation and urine samples for 8-isoPGF(2alpha) and 2,3-dinor 8-isoPGF(2alpha) assays were collected and blood pressures measured during the OGTT visit. Random plasma and urine samples were also obtained at 34-37 weeks. Glucose results were unblinded to the attending obstetrician if limits preset under the HAPO protocol were met. Maternal plasma 8-isoPGF(2alpha) and urinary 8-isoPGF(2alpha) and 2,3-dinor 8-isoPGF(2alpha) both at the time of OGTT (24-32 weeks) and at 34-37 weeks of gestation. Incidence of pre-eclampsia and gestational hypertension. Of the 408 women who attended for OGTT at 24-32 weeks, two met the glucose criteria for unblinding and 25 had missing 8-isoPGF(2alpha) values and thus were excluded from analysis. Of the 381 women, 338 (88.7%) attended for random plasma samples at 34-37 weeks. Significant correlations were observed between maternal fasting plasma isoprostane and both fasting (r= 0.20; P < 0.001) and 2-hour (r= 0.39; P < 0.001) plasma glucose levels at the time of OGTT. Gestational hypertension/pre-eclampsia occurred in 17 (4.2%) women, and at the time of OGTT, they had significantly higher fasting plasma 8-isoPGF(2alpha) (P < 0.001), urine 8-isoPGF(2alpha) (P < 0.005) and urine 2,3-dinor 8-isoPGF(2alpha) to creatinine ratios (P < 0.001), as well as higher MAP (P < 0.001) than women who remained normotensive. At 34-37 weeks, only random plasma 8-isoPGF(2alpha) was significantly higher (P < 0.001) among the women with gestational hypertension/pre-eclampsia. Plasma markers of oxidative stress were positively correlated with plasma glucose at the time of OGTT (24-32 weeks). Women who subsequently developed gestational hypertension/pre-eclampsia had significantly higher plasma and urine markers of oxidative stress at the time of OGTT but only higher plasma markers at 34-37 weeks. Plasma 8-isoPGF(2alpha) appears to be a very good predictor of subsequent gestational hypertension/pre-eclampsia when measured at the time of OGTT, but its ability to discriminate deteriorates as pregnancy advances.

Effect of short-term fasting on urinary excretion of primary lipid peroxidation products and on markers of oxidative DNA damage in healthy women

The goal of this study was to determine whether short-term fasting changes in urinary biomarkers related to oxidative stress: malondialdehyde (MDA), 8-isoprostaglandin F2alpha (8-isoPGF), 8-hydroxydeoxy-guanosine (8-OHdG) and 1,N6-ethenodeoxyadenosine (epsilondA) among female volunteers participating in the short-term fasting program in South Korea. The study subjects were 52 healthy women (mean age 28, range 15-48 years old) who provided urine samples both before and after the fasting program (average 7.2, range: 3-11 days). Urinary MDA was measured by HPLC-UV and epsilondA levels were measured by immuno-affinity purification followed by HPLC-fluorescence detection. Urinary 8-OHdG and 8-isoPGF concentrations were determined by ELISA. Plasma leptin levels were also measured by radioimmunoassay. Information on demographic characteristics, personal habits (smoking and alcohol consumption) and previous medical history were collected by a self-administered questionnaire. Percent loss of body weight (average 6.3%, 4.28 +/- 0.25 kg) was significantly correlated with fasting duration (r = 0.70, n = 52, P < 0.01). The plasma leptin levels after fasting (5.89 +/- 1.10 ng/ml) were significantly lower than before fasting (6.91 +/- 1.13 ng/ml) (n = 27, P = 0.05). Urinary MDA levels after fasting (0.18 +/- 1.10 mg/g creatinine) were significantly lower than before fasting (0.37 +/- 1.11) (n = 51, P < 0.01). Urinary 8-isoPGF also were significantly reduced after fasting (n = 47, P < 0.01). However, there was no significant difference in 8-OHdG or epsilondA. There was a statistically significant correlation between % change of urinary MDA level with % change of 8-isoPGF level (partial correlation coefficient r = 0.57, n = 46, P = 0.01). The correlations between % change of 8-OHdG and plasma leptin was also significant (partial correlation coefficient r = 0.51, n = 27, P = 0.02). Our results demonstrate that the short-term fasting reduces lipid peroxidation products but does not affect oxidative stress-induced DNA damage.

A thromboxane effect of a hydroxytyrosol-rich olive oil wastewater extract in patients with uncomplicated type I diabetes

To assess the antioxidant/non-antioxidant effects of a hydroxytyrosol (HT)-rich phenolic extract from olive mill wastewaters administered with a breakfast. Five type I diabetic patients received 25 mg of HT the first day and 12.5 mg/day the following 3 days. Blood sampling was carried out at T(0) (baseline) and T(4d) just before the breakfast + HT administration and at time points 1, 2, 3 and 4 h after T(0). Urines (24-h) were collected from T(0) to T(4d). Baseline HbA1c was generally inferior to 10%, glycemia was within the range 6-24 mmol/l, whereas total cholesterol, HDL-chol and triglycerides were normal. The major finding was the 46% decrease in the serum TXB(2) production after blood clotting at T(4d). Plasma vitamin A, E, beta-carotene were not changed. Vitamin C tended to increase (P = 0.075). Plasma antioxidant capacity was enhanced at T(0)+1 h only, whereas its main determinants (albumin, bilirubin, uric acid) were not modified. Urinary 8-isoPGF(2alpha) levels were highly variable and were not affected significantly by HT administration. The major effect of HT accounts for an antiaggregating platelet action, leading to a possible prevention of thrombotic and microthrombotic processes.

Increased urinary F 2-isoprostanes levels in the patients with Alzheimer’s disease

Isoprostanes, novel markers of oxidative injury, are generated by the free radical-mediated peroxidation of arachidonic acid (AA). They are thought to be important in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD). Using gas chromatography–mass spectrometry (GC–MS), we investigated the alteration of urinary F 2-isoprostanes in AD patients compared to that of healthy subjects. Our results show that the levels of urinary F 2-isoprostanes, sum of the prostaglandin F 2α isomer; prostaglandin F 2α (PGF 2α), prostaglandin F 2β (PGF 2β), and 8-isoprostaglandin F 2α (8-isoPGF 2α), significantly increased in AD patients ( P < 0.05). The concentration of urinary 8-isoPGF 2α, one of the biomarkers of oxidative stress, was not significantly different between 34 AD patients and 20 age-matched controls ( P > 0.05). The PGF 2α, formed by endoperoxide reductase from PGH 2, was significantly increased in AD patients, when compared with the levels of the normal controls ( P < 0.05). The PGF 2α, an enzymatic product of arachidonic acid, may affect the pathogenesis of AD. In addition, urinary F 2-isoprostanes can play an important role as a biomarker in AD.

Elevated urinary 8-isoprostaglandin F 2α in females with Graves’ hyperthyroidism

In this study, we investigate how oxidative stress alters the urinary F 2-isoprostanes’ level and we examine the correlation between 8-isoPGF 2α and thyroid hormones in female patients with Graves’ hyperthyroidism. We quantitatively determined the concentrations of urinary F 2-isoprostanes using gas chromatography-mass spectrometry in the selected ion-monitoring mode. We recruited individuals in the following three groups of subjects for this study: (1) female hyperthyroidism patients ( n=14, 21–71 years), (2) female hypothyroidism patients ( n=16, 16–66 years), and (3) female age-matched normal controls ( n=10, 20–61 years). The average concentration of 8-iso prostaglandin F 2α (8-isoPGF 2α) in hyperthyroidism patients was significantly higher than that in the healthy controls ( P<0.05). The concentration of 8-isoPGF 2α in hypothyroidism patients was similar to that in normal controls. Although the level of 8-isoPGF 2α in two of the patients was slightly elevated, the P value was not significant (NS). Our data suggest that the increased level of urinary 8-isoPGF 2α may reflect possible oxidation injuries in hyperthyroidism patients due to the high level of thyroid hormones. Therefore, the elevated 8-isoPGF 2α in patients could be used as an important biomarker for hyperthyroidism disease.

Urine 8-isoprostane F 2α concentrations in patients with neurogenic bladder due to spinal cord injury

Background: Isoprostanes are prostaglandin-like end products of arachidonic acid peroxidation that are produced by a free radical-catalyzed mechanism. Considering its free radical-dependent formation and potent contractor effect, it is postulated that isoprostane 8-iso PGF 2α may play an important role in oxidative stress-related smooth muscle dysfunction. These substances may also influence bladder activity directly by effects on the smooth muscle. The present study was designed to measure traditional biochemical parameters (MDA, TAS, vitamin E) in plasma and 8-iso PGF 2α concentrations in urine of patients with spinal cord injury and to evaluate the relation of urinary isoprostane concentrations to the bladder function. Methods: All spinal cord patients underwent urodynamic evaluations. The biochemical tests were performed in both hyperreflexic bladder group ( n=23) and areflexic bladder group ( n=10), and the findings were compared to those of the patients with normally functioning bladder (controls, n=19). Results: Urine 8-iso PGF 2α concentrations were significantly increased in hyperreflexic group (median value 0.89 pg/mg creatinine) compared to both control (0.52 pg/mg creatinine) and areflexic groups ( p<0.001). The lowest concentrations of urinary 8-iso PGF 2α were observed in the areflexic group (0.22 pg/mg creatinine), and these were positively correlated to the plasma MDA concentrations in areflexic patients ( p=0.05; r=0.684). Conclusion: Isoprostanes may be involved in the pathogenesis of neurogenic bladder dysfunction. It may be of value to determine the urinary concentrations of 8-iso PGF 2α in order to distinguish areflexic bladders from the hyperreflectics.