Measurement of Enzymatic and Nonenzymatic Polyunsaturated Fatty Acid Oxidation Products in Plasma and Urine of Macular Degeneration Using LC-QTOF-MS/MS.

Oxidized polyunsaturated fatty acids (PUFA) are associated to pathogenesis of diseases including cardiovascular and neurodegeneration. The novel products are not only biomarkers but also lipid mediators in gene regulation and signaling pathways. Herein, simultaneous quantitation of 28 products derived from nonenzymatic and enzymatic oxidation of PUFA i.e. 5-, 15-F2t -isoprostanes, 7-, 17-F2t -dihomo-isoprostanes, 7-, 17-F2t -dihomo-isofurans, 5-, 8-, 18-F3t -isoprostanes, 4-, 10-, 13-, 14-, 20-F4t -neuroprostanes, 5-, 8-, 9-, 11-,12-, 15-, 20-HETE, 4-, 7-, 11-, 14-, 17-HDHA, RvE1, and NPD1 using LC-(ESI)-QTOF-MS/MS was developed. These products were measurable in a single sample and the analytical time was relative short (~15 min). Furthermore, we showed that the use of internal standards is a requisite to normalize matrix effects and preparation loss for the quantitation. Validation assays indicated the method to be robust for plasma and mid-stream urine sample analysis in particular from those of age-related macular degeneration subjects, where the accuracy of quantitation displayed good repeatability.

Effects of Cyclic Fatty Acid Monomers from Heated Vegetable Oil on Markers of Inflammation and Oxidative Stress in Male Wistar Rats.

This study assesses the effects of cyclic fatty acid monomers (CFAM) from heated vegetable oils on oxidative stress and inflammation. Wistar rats were fed either of these four diets for 28 days: canola oil (CO), canola oil and 0.5% CFAM (CC), soybean oil (SO), and soybean oil and 0.5% CFAM (SC). Markers of oxidative stress and inflammation were determined by micro liquid chromatography tandem mass spectrometry (micro-LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) kits, respectively. Analysis of variance (ANOVA) for a 2 × 2 factorial design was performed to determine the CFAM and oil effects and interactions between these two factors at P ≤ 0.05. For significant interactions, a post hoc multiple comparison test was performed, i.e., Tukey HSD (honest significant difference) test. CFAM induced higher plasma levels of 15-F2t-IsoP (CC, 396 ± 43 ng/mL, SC, 465 ± 75 ng/mL vs CO, 261 ± 23 ng/mL and SO, 288 ± 35 ng/mL, P < 0.05). Rats fed the SC diet had higher plasma 2,3-dinor-15-F2t-IsoP (SC, 145 ± 9 ng/mL vs CC, 84 ± 8 ng/mL, CO, 12 ± 1 ng/mL, and SO, 12 ± 1 ng/mL, P < 0.05), urinary 2,3-dinor-15-F2t-IsoP (SC, 117 ± 12 ng/mL vs CC, 67 ± 13 ng/mL, CO, 15 ± 2 ng/mL, and SO, 18 ± 4 ng/mL, P < 0.05), and plasma IL-6 (SC, 57 ± 10 pg/mL vs CC, 48 ± 11 pg/mL, CO, 46 ± 9 pg/mL, and SO, 44 ± 4 pg/mL, P < 0.05) than the other three diet groups. These results indicate that CFAM increased the levels of markers of oxidative stress, and those effects are exacerbated by a CFAM-high-linoleic acid diet.

Reliable determination of new lipid peroxidation compounds as potential early Alzheimer Disease biomarkers.

Lipid peroxidation plays an important role in Alzheimer Disease, so corresponding metabolites found in urine samples could be potential biomarkers. The aim of this work is to develop a reliable ultra-performance liquid chromatography-tandem mass spectrometry analytical method to determine a new set of lipid peroxidation compounds in urine samples. Excellent sensitivity was achieved with limits of detection between 0.08 and 17 nmol L-1, which renders this method suitable to monitor analytes concentrations in real samples. The method's precision was satisfactory with coefficients of variation around 5-17% (intra-day) and 8-19% (inter-day). The accuracy of the method was assessed by analysis of spiked urine samples obtaining recoveries between 70% and 120% for most of the analytes. The utility of the described method was tested by analyzing urine samples from patients early diagnosed with mild cognitive impairment or mild dementia Alzheimer Disease following the clinical standard criteria. As preliminary results, some analytes (17(RS)-10-epi-SC-Δ15-11-dihomo-IsoF, PGE2) and total parameters (Neuroprostanes, Isoprostanes, Isofurans) show differences between the control and the clinical groups. So, these analytes could be potential early Alzheimer Disease biomarkers assessing the patients' pro-oxidant condition.

GC-MS Analysis of Lipid Oxidation Products in Blood, Urine, and Tissue Samples.

Oxidant stress has been identified as important in the pathology of many diseases. Oxidation products of polyunsaturated fatty acids collectively termed isoprostanes, neuroprostanes, and isofurans are considered the most reliable measures of in vivo lipid oxidation, and they are widely used to assess oxidant stress in various diseases. Here we describe the measurement of these lipid oxidation products using gas chromatography mass spectrometry with electron capture negative ionization.

Potential applications of lipid peroxidation products - F4-neuroprostanes, F3-neuroprostanesn-6 DPA, F2-dihomo-isoprostanes and F2-isoprostanes - in the evaluation of the allograft function in renal transplantation.

F4-neuroprostanes, F3-neuroprostanesn-6 DPA, and F2-dihomo-isoprostanes, metabolites of non-enzymatic lipid peroxidation of polyunsaturated fatty acids [docosahexaenoic acid, n-6 docosapentanoic acid, and adrenic acid respectively], have become important biomarkers for oxidative stress in several diseases like epilepsy and alzheimer. These biomarkers and the 15-F2t-isoprostane (also known as 8-iso-PGF2α), a F2-isoprostane isomer measured as reference oxidative marker at systemic level, were analyzed by UHPLC-QqQ-MS/MS in the urine of 60 renal recipients from cadaveric donors of the Nephrology Unit of the University Hospital Virgen de la Arrixaca, at six different times during the first six months after renal transplantation, and were compared with a control group of 60 healthy subjects from the same hospital. A total of 11 metabolites were analyzed and different patterns were observed. A tendency to decrease was observed in three metabolites (4-epi-4-F3t- NeuroPn-6 DPA, ent-7(RS)-7-F2t-dihomo-IsoP, and ent-7(S)-7-F2t-dihomo-IsoP) and in our reference oxidative marker (15-F2t-IsoP) when kidney function improved and the excretion of urine proteins decreased. These results suggest that these three biomarkers of oxidative stress could be useful to assess renal function in the postransplant phase. Unfortunately, little is known about this kind of biomarker in this cohort of patients, so further investigation would be required in the clinical field to clarify the relationship between oxidative stress and the graft function, as well as the usefulness of these biomarkers as rejection markers.

Assessment of oxidative stress biomarkers - neuroprostanes and dihomo-isoprostanes - in the urine of elite triathletes after two weeks of moderate-altitude training.

This randomized and controlled trial investigated whether the increase in elite training at different altitudes altered the oxidative stress biomarkers of the nervous system. This is the first study to investigate four F4-neuroprostanes (F4-NeuroPs) and four F2-dihomo-isoprostanes (F2-dihomo-IsoPs) quantified in 24-h urine. The quantification was carried out by ultra high pressure liquid chromatography-triple quadrupole-tandem mass spectrometry (UHPLC-QqQ-MS/MS). Sixteen elite triathletes agreed to participate in the project. They were randomized in two groups, a group submitted to altitude training (AT, n = 8) and a group submitted to sea level training (SLT) (n = 8), with a control group (Cg) of non-athletes (n = 8). After the experimental period, the AT group triathletes gave significant data: 17-epi-17-F2t-dihomo-IsoP (from 5.2 ± 1.4 µg/mL 24 h(-1) to 6.6 ± 0.6 µg/mL 24 h(-1)), ent-7(RS)-7-F2t-dihomo-IsoP (from 6.6 ± 1.7 µg/mL 24 h(-1) to 8.6 ± 0.9 µg/mL 24 h(-1)), and ent-7-epi-7-F2t-dihomo-IsoP (from 8.4 ± 2.2 µg/mL 24 h(-1) to 11.3 ± 1.8 µg/mL 24 h(-1)) increased, while, of the neuronal degeneration-related compounds, only 10-epi-10-F4t-NeuroP (8.4 ± 1.7 µg/mL 24 h(-1)) and 10-F4t-NeuroP (5.2 ± 2.9 µg/mL 24 h(-1)) were detected in this group. For the Cg and SLT groups, no significant changes had occurred at the end of the two-week experimental period. Therefore, and as the main conclusion, the training at moderate altitude increased the F4-NeuroPs- and F2-dihomo-isoPs-related oxidative damage of the central nervous system compared to similar training at sea level.