WO2017027976A1

WO2017027976A1 - (±)5-8,12-isoprostane class vi as a marker for early prediction of pre-eclampsia

Info

Publication number: WO2017027976A1
Application number: PCT/CA2016/050971
Authority: WO
Inventors: Jean-François BILODEAU; Pierre Julien
Original assignee: UNIVERSITé LAVAL
Priority date: 2015-08-20
Filing date: 2016-08-19
Publication date: 2017-02-23

Abstract

The present invention relates to a method of measuring level of (±)5-8,12-isoprostane class VI, as an early predictive marker of pre-eclampsia (PE). The present invention also relates to measuring the level of this marker and particularly establishing a ratio over other parameters such as polyunsaturated fatty acids (PUPA) or antioxidants. In particular, the method determines the levels of (±)5-8,12-isoprostane class VI in a biological sample from a pregnant woman prio ot the appearance of symptoms of PE.

Description

(±)5-8, 12-ISOPROSTANE CLASS VI AS A MARKER FOR EARLY PREDICTION OF PRE-ECLAMPSIA

Field of the invention

[0001] The present invention relates to a methodology for measuring isoprostanes profile in a human biological sample, particularly from pregnant women. The level of a particular regioisomer of isoprostane class VI and its ratio with certain other blood fatty acids allows one to determine the risk of a pregnant woman to develop pre-eclampsia early during pregnancy, much before the appearance of first symptoms.

Background of the Invention [0002] Pre-eclampsia (PE) affects approximately 3-5% of all pregnancies and is a leading cause of maternal death in North America and the UK. This disease, or the threat of onset, is the commonest cause of elective premature delivery, accounting for approximately 15% of all premature births. PE is defined according to the guidelines of the International Society for the Study of Hypertension in Pregnancy and includes amongst other factors: gestational hypertension and proteinuria. Gestational hypertension is defined as two recordings of diastolic blood pressure of 90 mm Hg or higher at least 4 h apart, and severe pressure of 1 10 mm Hg or higher at least 4 h apart or one recording of diastolic blood pressure of at least 120 mm Hg. Proteinuria is defined as excretion of 300 mg or more in 24 h or two readings of 2+ or higher on dipstick analysis of midstream or catheter urine specimens if no 24 h collection was available.

[0003] Women are classified as previously normotensive or with chronic hypertension before 20 weeks' gestation. For previously normotensive women, PE is defined as gestational hypertension with proteinuria and severe PE as severe gestational hypertension with proteinuria. For women with chronic hypertension, superimposed PE is defined by the new development of proteinuria. The measurement of blood pressure and testing for proteinuria in all pregnant women is carried out predominantly for the detection of PE. These procedures and the care of affected women and of the premature children make considerable demands on healthcare resources. [0004] There is no widely accepted or accurate method for the early prediction of PE. Elevation of the blood pressure and detection of protein in the urine occur when the disease process is well established. Detection of an abnormality of the blood flow to the uterine artery by Doppler ultrasound in women who later develop PE has been of some predictive use but this abnormality has been found to be relatively non-specific and for this reason has not been adopted in routine clinical practice.

[0005] Although some plasma/urine biochemical markers have been shown to be abnormal in the disease process, no single marker has proven to be of adequate sensitivity for use as a predictive indicator. For example, the use of placenta growth factor (PIGF) alone as a predictive indicator of PE has been proposed, but the predictive power of this marker could not be determined with any certainty. For example, international patent application WO 98/28006 suggests detecting PIGF alone or in combination with vascular endothelial growth factor (VEGF) in order to predict the development of PE.

[0006] US 5,891 ,622 teaches that isoprostanes are used to quantify an oxidative stress associated to numerous pathologies. This patent is focused on the use of ELISAs to measure free, conjugated or esterified isoprostanes (IsoPs) at large in the plasma, urine, cerebrospinal, bile and joint fluids. Alternatively, GC/MS can be used for the determination of IsoPs.

[0007] US patent 7,833,795 describes a method to assess cardiovascular risk using isoprostanes and liquid chromatography/tandem mass spectrometry in urine and plasma exclusively. Although, it is true that PE increases the risk of being affected by cardiovascular diseases later in life, PE is not a cardiovascular disease per se. The focus of the patent is on three isomers: 8,12-iso-iPF_2a-VI, 8-iso-PGF_2a, and iPF_2a-VI . According to the inventors, additional parameters are required to predict cardiovascular risk and comprise: thromboxane metabolites and a PGI₂ metabolite in urine, blood pressure, blood level of C-reactive protein, blood level of interleukin-6 (IL-6), blood level of soluble intracellular adhesion molecule-1 (slCAM-1 ), blood level of monocyte chemoattractant protein-1 (MCP-1 ), blood level of homocysteine, presence or extent of atherosclerotic plaques, and presence of one or more genetic predispositions for elevated cardiovascular risk. [0008] Although, there is no widely used treatment for PE (other than premature delivery), Chappell et al. [ref. 1 ] have shown a significant reduction in PE in high-risk women given supplements of vitamin C and vitamin E. In this study, risk was assessed by a test of relatively low sensitivity. More accurate and robust identification of women at risk would target those women most likely to benefit from this, or alternative, prophylactic therapies. Those identified at lower risk could be provided with less intensive and less expensive antenatal care.

[0009] Despite previous encouraging results of antioxidant vitamin trials, vitamin C and E supplementation did not reduce the rate of PE, but increased the risk of fetal loss or perinatal death and preterm pre-labor rupture of membranes in a large Canadian cohort [ref. 2]. Indeed, other antioxidants need to be investigated.

[0010] More recently, WO 2013/170369 disclosed the combined measure of iPF_2a-VI and 5 iPF_2a-VI for pre-eclampsia prediction, and various ratios of these isomers with fatty acids, to constitute as many tools for the early prediction of pre- eclampsia before the trigger of symptoms.

[0011] We have now found another isoprostane isomer that constitutes a clearly superior biomarker to the prior art.

[0012] Isoprostane separation is very challenging since there are 64 possible isomers derived from the oxidation of the arachidonic acid. These isomers are further subclassified in four series or class of regioisomers of 16 members each denoted as 5- , 12-, 8-, and 15-series or class III, IV, V and VI according to Taber's and Rokach's nomenclatures respectively [ref. 16, 17]. The classes of isomers can be further differentiated by MS/MS fragmentation using specific transition. However, up to now, regioisomers of a given class could only be separated by GC and, more recently, by HPLC in plasma samples from pregnant women [ref. 15].

Summary of the Invention

[0013] It has now been found that a specific isoprostane marker provides the much needed predictive parameter for a desired early prediction and/or diagnosis of PE. This marker is 5-8,12 -isoprostane class VI. Optionally the marker's total level is measured in combination with plasma volume, body mass index, antioxidant levels or blood lipid profile to provide a ratio predictive of early onset of PE.

[001 ] Therefore, in a first aspect, there is provided a method for measuring (±)5- 8,12-isoprostane class-VI in a human subject, comprising the steps of: a) obtaining a biological sample from the patient; b) extracting lipids from the sample; c) separating the (±)5-8,12-isoprostane class-VI [(±)5-8,12-iso-iPF2a-VI] from the extracted lipids; and d) measuring a level of isoprostane (±)5-8,12-iso-iPF2a-VI.

[0015] According to a second aspect, there is provided a method for measuring (±)5-8,12-isoprostane class-VI in a human subject, comprising the steps of: a) obtaining a biological sample from the patient; b) extracting lipids from the sample; c) measuring a level of (±)5-8,12-isoprostane class-VI [(±)5-8,12-iso-iPF2a-VI] with Laser Diode Thermal Desorption coupled to mass spectrometry (LDTD-MS).

[0016] In accordance with a further aspect, there is provided the method as defined above wherein the subject is a pregnant woman, this method further comprising: comparing the level from step d) with a control level from a control population representative of the pregnant woman; and reporting the comparison to the pregnant woman's treating physician; whereby when the level is at least about 15% higher than the control level, the physician may diagnose pre-eclampsia and, optionally take measures to monitor or treat the pregnant woman. [0017] In accordance with a further aspect, there is provided the method as defined above wherein the subject is a pregnant woman, and the method further optionally comprises: e) measuring from the sample a parameter selected from:

plasma volume, antioxidant level, body mass index and blood lipid profile; f) establishing a ratio of (±)5-8,12-iso-iPF2a-VI over one of the parameter from step e) for the subject; g) comparing the ratio from step f) with a control ratio from a control population representative of the pregnant woman; h) reporting the comparison from step g) to the pregnant woman's treating physician; whereby when the level or ratio is at least about 15% higher than the control level or control ratio, the physician may diagnose pre-eclampsia and, optionally take measures to monitor or treat the pregnant woman. [0018] In accordance with a further aspect, there is provided a method for predicting the appearance of preeclampsia (PE) in a pregnant woman comprising the steps of: a) obtaining a sample from the pregnant woman; b) measuring a level of isoprostane (±)5-8,12-iso-iPF2a-VI in the sample; c) comparing the level with a control level for a population or an individual representative of the pregnant woman; and d) determining if the comparing of step c) is higher than about 15% of the control level; and e) optionally reporting the determination from step f) to the subject's treating physician.

[0019] According to a particular aspect, the method described above can further comprises step: b') assessing at least one parameter selected from: plasma volume, body mass index and blood lipid profile in the sample; and b") establishing a ratio of isoprostane (±)5-8,12-iso-iPF2a-VI over at least one of a parameter selected from the group consisting of: plasma volume, body mass index and blood lipid profile from the sample; c') comparing the ratio with a control ratio for a population or an individual representative of the pregnant woman; and d') determining if the comparing of step c') is higher than about 15% of the control ratio; and e') optionally reporting the

determination from step f) to the subject's treating physician.

[0020] According to a further aspect, there is provided a method for treating a pregnant woman at risk of developing pre-eclampsia comprising: a) measuring (±)5- 8,12-isoprostane class-VI from a sample in accordance with the method above; b) determining that the ratio is at least above 15% of a control ratio; and c) administering an effective amount of a lipid selected from: AA, DHA, ω-3 fatty acids; a-tocopherol; v- tocopherol, vitamin E, CoQ10; and β-carotene to the pregnant woman to reach a ratio corresponding to a ratio of a control population; wherein the control population is a population of pregnant women characterized by normal blood pressure and absence of: obesity, diabetes, urine protein excretion and medical complications.

[0021] A further aspect of the present invention is to provide a method for measuring blood isoprostane profile of a pregnant woman, comprising the steps of: extracting lipids from a biological sample of the pregnant woman; separating 5-8,12 - isoprostane class VI (5-8,12-iPF_2a-VI), or derivatives thereof, from the lipids by chromatography; and measuring level of the 5-8,12 -iPF_2a-VI by mass spectrometry. [0022] Still, a further aspect of the present invention is to provide a method for measuring blood isoprostane profile of a pregnant woman, comprising the steps of: extracting lipids from a blood-derived sample of the pregnant woman; measuring a level of 5-8,12-iPF_2a-VI or a derivative thereof; and comparing the level with a control level from a control population representative of the pregnant woman; wherein when the level or ratio is at least about 15% higher than the control level or ratio, the level is reported to a treating physician. Optionally, the physician may diagnose pre-eclampsia or risk of developing pre-eclampsia and, optionally may take measures to monitor or treat the pregnant woman. [0023] Therefore, in a further aspect, the present invention provides a method for measuring 5-8,12 iso-iPF_2a-VI in a pregnant woman, comprising the steps of: a) obtaining a sample from the pregnant woman; b) extracting lipids from the sample; c) measuring levels of 5-8,12 iso-iPF_2a-VI; d) measuring at least one of parameters selected from: plasma volume, antioxidant level, body mass index and blood lipid profile from the sample; d) optionally, establishing a ratio of 5-8,12 iso-iPF_2a-VI over at least one of the parameters for the pregnant woman; and e) comparing the amount or the ratio with a control level or ratio from a control population or individual representative of the pregnant woman; and f) optionally, reporting the comparison from step e) to the subject's treating physician; wherein when the level or ratio is at least about 15% higher than the control level or ratio, the physician may diagnose preeclampsia and, optionally take measures to monitor or treat the pregnant woman. Particularly, the pregnant woman is at risk of developing pre-eclampsia.

[0024] In accordance with an aspect of the invention, there is provided a method of specific prediction of PE in a subject, comprising determining in a maternal biological sample a level of a 5-8,12 iso-iPF_2a-VI, wherein the amount of 5-8,12 iso-iPF_2a-VI above a control level is indicative that the subject is at risk of developing PE.

[0025] A further aspect of the present invention provides a method of specific prediction of PE in a pregnant woman, comprising determining in a maternal sample a level of isoprostane 5-8,12 iso-iPF_2a-VI , wherein the amount of a 5-8,12 iso-iPF_2a-VI above a control level is indicative that the woman is at risk of developing PE. [0026] An alternative aspect of the present invention provides a method of specific prediction of PE in a pregnant woman, comprising determining in a maternal sample the ratio of 5-8,12 iso-iPF_2a-VI over polyunsaturated fatty acids (PUFA), wherein a higher ratio is indicative that the pregnant woman is at risk of developing PE. [0027] The present invention provides a method of specific prediction of PE in a pregnant woman, comprising determining in a maternal sample the ratio a 5-8,12 iso- iPF_2a-VI over omega-3 and/or omega-6 polyunsaturated fatty acids (PUFA), wherein a higher ratio is indicative that the pregnant woman is at risk of developing PE.

[0028] Optionally, the method also comprises the additional step of taking measures to place this woman under surveillance or tight monitoring, and/or adjusting antioxidant intake.

[0029] It has been found that by measuring the markers or ratios mentioned above, it is possible to determine with high specificity and sensitivity whether a subject is likely to develop PE. [0030] Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims.

Description of the invention

Brief Description of the Drawings

[0031] Having thus generally described the aspects of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, particular embodiments thereof, and in which the figures represent:

[0032] FIG. 1. HPLC gradient used for the analysis of F₂-isoprostanes. Solvent A : H₂0 + 0.01 % acetic acid; solvent B : ACN + 0.01 % acetic acid; Solvent C : MeOH + 0.01 % acetic acid.

[0033] FIG. 2. Representation of the structures of F₂-isoprostanes used to setup the HPLC-MS-MS method. [0034] FIG. 3. Representation of the structure of the deuterated F₂-isoprostane internal standards used to setup the HPLC-MS-MS method.

[0035] FIG. 4: Comparison between conventional HPLC-MS/MS (A) and three- dimensional separation of isoprostanes of class VI using the combination of HPLC, ion mobility and tandem mass spectrometry (MS/MS) (B) . Plasma sample was spiked with 0.1 ng of isoprostane standards. Peak identification: A: iPF2a-VI ; B: 5-iPF2a-VI ; C: 5- 8,12-iso-iPF2a-VI.

[0036] FIG. 5. Receiver operating characteristic (ROC) curves for isoprostane (iPF2a-VI + 5 iPF2a-VI) normalized to plasma volume from WO 2013/170369 ( ), (±)5-8,12-iso-iPF2a-VI normalized to plasma volume ( ) and the following equation taking into account factors that can influence isoprostane measurements: (±)5-8,12- iso-iPF2a-VI (pg) x BMI / (Ul ω-3 / (a-tocopherol /CoQ10 /p-carotene ))(- - - ). Omega-3 fatty acids (ω-3) = C18:3w3 + C18:4w3 + C20:3w3 + C20:4w3 + C20:5w3 + C22:5w3 + C22:6(JU3. Unsaturation index for omega-3 (Ul ω-3) = (%Monoenoic x 1 ) + (%Dienoic x 2) + (%Trienoic x 3) + (%Tetraenoic x 4) + (%Pentaenoic x 5) + (%Hexaenoic x 6) of ω-3 fatty acids.

[0037] FIG. 6 Lactone formation from (±)5-8,12-iso-iPF2a -VI under acidic conditions

[0038] FIG. 7. Example of a derivatization mechanism for F2-isoprostanes. From Milne, G. L, Sanchez, S. C, Musiek, E. S., and Morrow, J. D. (2007) Quantification of F2-isoprostanes as a biomarker of oxidative stress. Nat Protoc 2, 221 -226. PFBB=Pentafluorobenzyl bromide, DIPE=N,N'-Diisopropylethylamine, BSTFA=N,0- bis(trimethylsilyl).

[0039] FIG. 8. Detection of (±)5-8,12-iso-iPF2a-VI using LDTD technology. Detection is linear from 0.3 to 3000 pg/well (top panel). The analysis time is below 9 sec per sample (bottom panel: the internal standard in gray and the signal in black).

[0040] FIG. 9. Profile of F2-isoprostanes normalized to omega-3 and omega-6 ratio in control and preeclamptic pregnancies. ^*: different from the control group (p<0.05, T-test). Definitions and abbreviations

[0041] With respect to the invention presented herein, the following definitions and abbreviations are used, wherein:

[0042] The terms "5-8,12-iso-iPF₂a -VI", "(±)5-8,12-iso-iPF₂a-VI" and 8,12-iso- iPF₂a -VI as used herein indiscriminately, all mean between 1 to 4 possible isomers in theory.

[0043] The abbreviation "AA" means arachidonic acid.

[0044] The abbreviation "ΒΜ means body mass index as measured before pregnancy, according to health Canada definition (weight(kg)/height(m)²) (hc- sc.gc.ca/fn-an/nutrition/weights-poids/guide-ld-adult/index-eng.php).

[0045] The abbreviation "DHA" means docosahexaenoic acid.

[0046] The abbreviation "CoQ10" means coenzyme Q10.

[0047] The abbreviation "iP" means isoprostane, whereas the abbreviation iPF_2a means F_2a- isoprostane. [0048] The abbreviation "PUFA" means polyunsaturated fatty acids.

[0049] The abbreviation LDTD means Laser Diode Thermal Desorption

[0050] The term "pre-eclampsia" (PE) as used herein is defined according to the guidelines of the International Society for the Study of Hypertension in Pregnancy, as described above. [0051] The terms "biological sample" or "maternal sample" are taken from a human subject, particularly a pregnant woman and can be any sample from which it is possible to measure the markers mentioned herein. Particularly the sample is any tissue that contains lipids. More particularly, the sample is urine, saliva or blood- derived. For maternal samples, the samples can be taken at any time from about 10 weeks gestation. Preferably the sample is taken at between 12 and 24 weeks gestation, more preferably the sample is taken before 20 weeks. [0052] The term "sensitivity" is defined as the proportion of true positives (i.e. will develop PE) identified as positives in the method.

[0053] The term "specificity" is defined as the proportion of true negatives (i.e. will not develop PE) identified as negatives in the method. [0054] The term "specific prediction of pre-eclampsia" as used herein means that the method of the present invention is used to specifically predict the development of PE. In particular, the method of the present invention enables one to determine whether an individual is likely to develop PE.

[0055] The abbreviation "Ul" means unsaturation index and is calculated in the following manner: unsaturation index for omega-3 (Ul ω-3) = (%Monoenoic x 1 ) + (%Dienoic x 2) + (%Trienoic x 3) + (%Tetraenoic x 4) + (%Pentaenoic x 5) + (%Hexaenoic x 6) of fatty acids; or unsaturation index for omega-6 (Ul ω-6 ) = (%Monoenoic x 1 ) + (%Dienoic x 2) + (%Trienoic x 3) + (%Tetraenoic x 4) + (%Pentaenoic x 5) + (%Hexaenoic x 6) of fatty acids. [0056] The term control population means a population of pregnant women characterized by a normal blood pressure (≤140/90 mm Hg), and absence of: diabetes, obesity (BMI >30), urine protein excretion and medical complications.

Detailed description of particular embodiments

[0057] Applicant has obtained samples of blood from pregnant women who were considered at risk of PE on the basis of the uterine artery Doppler test or because they had had the disease in a previous pregnancy. Blood samples were obtained respectively twice from 12 to 18 weeks and 24 to 26 weeks of pregnancy. A selection of biochemical markers implicated in PE were measured, including vitamin C, homocysteine, plasma lipids and 8-epi prostaglandin F_2a but none proved to be effective in prediction. We found that the ratio of total isoprostanes over blood fatty acids increased prior to the onset of the disease. Combinations of these markers proved to be excellent in the sensitive and specific prediction of subsequent PE. Method

[0058] Therefore, in a first aspect, there is provided a method for measuring (±)5- 8,12-isoprostane class-VI in a human subject, comprising the steps of: a) obtaining a biological sample from the subject; b) extracting lipids from the sample; c) separating and measuring the (±)5-8,12-isoprostane class-VI [(±)5-8,12-iso-iPF2a-VI]. Particularly, the separating is carried out by gas, liquid or ion mobility chromatography, more particularly by HPLC. Still, particularly, the measuring is carried out by mass spectrometry (MS), even more particularly by MS coupled to an ion-mobility device.

[0059] The present invention therefore provides a method of specific prediction of PE in a subject comprising the steps of: a) determining in a maternal sample the level of 5-8,12 iso-iPF2a-VI ; b) determining in the maternal sample a parameter selected from the group consisting of: BMI, plasma volume, antioxidant levels and blood lipid profile; c) establishing a ratio of 5-8,12 iso-iPF2a-VI over at least one or the parameter; wherein the ratio above (or under) a predetermined ratio is indicative that the pregnant woman is at risk (or not, respectively) of developing PE.

[0060] The method in the present invention may be performed in conjunction with other tests for diagnostic indicators, such as blood pressure, level of uric acid, etc.

De vatization of isoprostane for detection and measurement

The method according to any one of claims 1 to 6, further comprising a step of: c') treating (±)5-8,12-iso-iPF_2a -VI under acidic conditions to produce 5, 8,12-iso-iPF2a-VI- 1 ,5-lactone; and d) measuring level of said lactone.

The method according to any one of claims 1 to 6, further comprising a step of: c') derivatizing (±)5-8,12-iso-iPF_2a -VI into a derivative thereof; and d) measuring a level of isoprostane (±)5-8,12-iso-iPF_2a-VI derivative thereof.

The method according to claim 8, wherein said derivative is selected from the group consisting of: pentafluorobenzyl ester, 4-diazomethylpyridine, 2-diethyl aminoethyl chloride, 2-diethylaminoethyl bromide, trimethylanilinium hydroxide, and 2,4- bis(diethylamino)-6-hydrazino-1 ,3,5-triazine. Ratio and control level

[0061] In order to determine whether the level or ratio of the markers referred to above is greater than normal, the normal level (i.e. control) or ratio of the relevant control population or individual needs to be determined. The relevant control population or individual may be defined based on, for example, ethnic background or any other characteristic that may affect normal levels of the marker. The relevant population or individual for establishing the normal level or ratio of the markers is preferably selected on the basis of low risk for PE (i.e. no known risk marker for PE, such as previous PE, diabetes, obesity, prior hypertension etc.). [0062] Particularly, the control population is a population of normotensive pregnant women characterized by a normal blood pressure (≤140/90 mm Hg), and absence of diabetes, obesity (BMI >30), urine protein excretion and medical complications.

[0063] Once the normal levels are known, the measured levels can be compared and the significance of the difference determined using standard statistical methods. If there is a statistically significant difference between the measured level and the normal level, then there is a significant risk that the individual from whom the levels have been measured will develop PE.

[0064] Particularly, there is a significant difference when the sample level is increased by at least about 15% compared to the control level, particularly at least about 15%, 20%, 25%, more particularly at least about 50%, 100%, 200%, 300% or even above 400%.

[0065] Of course, the present invention teaches that a certain ratio when higher than a control ratio is indicative of preeclamptic condition. Should a person skilled in the art decide to inverse the ratio taught (such as for example β-carotene over 5-8,12 iso-iPF₂a -VI), then a decreased ratio will lead to the same conclusion (see Table 2). The methods and assays as claimed also encompass this reverse ratio.

[0066] Still, more particularly, in step c) the method comprises the determination of a ratio selected from the group consisting of: (±)5-8,12-iso-iPF2a-VI (pg) / plasma volume (ml); (±)5-8,12-iso-iPF2a-VI (pg) x BMI (Kg/m²) / plasma volume (ml); (±)5- 8,12-iso-iPF2a-VI (pg) / a-tocopherol (μΜ); (±)5-8,12-iso-iPF2a-VI (pg) / γ-tocopherol (μΜ); (±)5-8,12-iso-iPF2a-VI (pg) / a-tocopherol / γ-tocopherol); (±)5-8,12-iso-iPF2a-VI (pg) / vitamin E (μΜ); (±)5-8,12-iso-iPF2a-VI (pg) / β-carotene (μΜ); (±)5-8,12-iso- iPF2a-VI (pg) / CoQ10 (μΜ); (±)5-8,12-iso-iPF2a-VI (pg) / (ω-3 / ω-6); (±)5-8,12-iso- iPF2a-VI (pg) / DHA (pg); (±)5-8,12-iso-iPF2a-VI (pg) / AA (pg); (±)5-8,12-iso-iPF2a-VI (pg) / % ω-3; (±)5-8,12-iso-iPF2a-VI (pg) ^* BMI (Kg/m2) / AA (μg); (±)5-8,12-iso-iPF2a- VI (pg) x BMI (Kg/m2) / DHA (μg); (±)5-8,12-iso-iPF2a-VI (pg) / (β-carotene / %ω-3); (±)5-8,12-iso-iPF2a-VI (pg) / (β-carotene / Ul ω-3 9); (±)5-8,12-iso-iPF2a-VI (pg) / (β- carotene / PI ω-3 10); (±)5-8,12-iso-iPF2a-VI (pg) / (Ul ω-3 / (CoQ10 / β-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) / (% ω-3 / (CoQ10 / ω-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) / (Ul ω-3/ (a-tocopherol / CoQ10 ^-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) / (% ω-3 / (a-tocopherol / CoQ10 ^-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) / (DHA / (a- tocopherol / CoQ10 ^-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) / (AA / (a-tocopherol / CoQ10 ^-carotene)); (±)5-8,12-iso-iPF2a-VI (pg) x BMI / (AA / (a-tocopherol / CoQ10 ^-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) x BMI / (DHA / (a-tocopherol / CoQ10 /β- carotene )); (±)5-8,12-iso-iPF2a-VI (pg) x BMI / (% ω-3 / (a-tocopherol / CoQ10 /β- carotene )); and (±)5-8,12-iso-iPF2a-VI (pg) x BMI / (Ul ω-3 / (a-tocopherol / CoQI O /β- carotene )); whereby when the ratio is above a control ratio is indicative that the pregnant woman is at risk of developing PE.

[0067] More particularly, the method comprises the establishment of a ratio selected from the group consisting of: (±)5-8,12-iso-iPF2a-VI (pg) / β-carotene (μΜ); (±)5-8,12-iso-iPF2a-VI (pg) / (β-carotene / % ω-3); (±)5-8,12-iso-iPF2a-VI (pg) / (β- carotene / Ul ω-3); (±)5-8,12-iso-iPF2a-VI (pg) / (β-carotene / PI ω-3); (±)5-8,12-iso- iPF2a-VI (pg) / (Ul ω-3 / (CoQ10 / β-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) / (% ω-3 / (CoQ10 / β-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) / (Ul ω-3/ (a-tocopherol /CoQ10 /β- carotene )); (±)5-8,12-iso-iPF2a-VI (pg) / (% ω-3 / (a-tocopherol /CoQ10 ^-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) / (DHA / (a-tocopherol /CoQ10 ^-carotene )); (±)5-8,12-iso- iPF2a-VI (pg) / (AA / (a-tocopherol /CoQ10 ^-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) x BMI / (AA / (a-tocopherol /CoQ10 ^-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) x BMI / (DHA / (a-tocopherol /CoQ10 ^-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) x BMI / (% ω-3 / (a-tocopherol /CoQ10 ^-carotene )); and (±)5-8,12-iso-iPF2a-VI (pg) x BMI / (Ul ω-3 / (a-tocopherol /CoQ10 ^-carotene )). [0068] Finally, even most particularly, in step c) the method comprises the determination of a ratio calculated as: (±)5-8,12-iso-iPF2a-VI (pg) x BMI over (Ul ω-3 / (γ-tocopherol /CoQ10 ^-carotene)).

[0069] Particularly, there is a significant difference when the ratio is increased by at least about 15% compared to the control level, particularly at least about 20%, 25%, more particularly at least about 50%, 100%, 200%, 300% or even above 400%.

[0070] It can be seen that the level of sensitivity and specificity can be altered by altering the control level. In some situations, e.g. when screening large numbers of women at low risk of PE, it is important to have high specificity. In other situations, it may be important to have a balance between high sensitivity and specificity, e.g. when considering individual women at high risk of PE a balance between high sensitivity and specificity is needed.

Assay

[0071] In accordance with a particular aspect, the invention provides an assay for predicting the appearance of preeclampsia (PE) in a pregnant woman comprising the steps of: a) obtaining a sample from the pregnant woman; b) assessing a level of isoprostane (±)5-8,12-iso-iPF2a-VI in the sample; c) comparing the level with a control level for a population or an individual representative of the pregnant woman; and d) determining if the comparing of step c) is higher than about 10% of the control level; and e) optionally reporting the determination from step f) to the subject's treating physician.

[0072] According to an alternative aspect, this assay further comprises the step of: b') assessing at least one parameter selected from: plasma volume, body mass index, antioxidant levels and blood lipid profile in the sample; and b") establishing a ratio of isoprostane (±)5-8,12-iso-iPF2a-VI over at least one of a parameter selected from the group consisting of: plasma volume, body mass index, antioxidant levels and blood lipid profile from the sample; c') comparing the ratio with a control ratio for a population or an individual representative of the pregnant woman; and d') determining if the comparing of step c') is higher than about 15% of the control ratio; and e') optionally reporting the determination from step f) to the subject's treating physician. [0073] Alternatively, the control level or ratio of this assay is established with a control population or individual, wherein the control population or individual is selected from the group consisting of: an individual in a normal population devoid of PE symptoms; a non-pregnant woman; same pregnant subject prior to pregnancy; and same pregnant subject prior to 10 weeks of pregnancy.

[0074] In accordance with another aspect, the invention provides the assay as defined above, wherein the level or the ratio is increased by at least about 15%, 20%, 25%, 50%, 100%, 200%, 300% or even greater than 400%.

[0075] According to an alternative aspect of the present invention, there is provided the assay as defined above, wherein the ratio is selected from a group consisting of: (±)5-8,12-iso-iPF2a-VI (pg) / plasma volume (ml); (±)5-8,12-iso-iPF2a- VI (pg) x BMI 4 (Kg/m2) / plasma volume (ml); (±)5-8,12-iso-iPF2a-VI (pg) / a- tocopherol (μΜ); (±)5-8,12-iso-iPF2a-VI (pg) / γ-tocopherol (μΜ); (±)5-8,12-iso- iPF2a-VI (pg) / a-tocopherol / γ-tocopherol); (±)5-8,12-iso-iPF2a-VI (pg) / vitamin E (μΜ); (±)5-8,12-iso-iPF2a-VI (pg) / β-carotene (μΜ); (±)5-8,12-iso-iPF2a-VI (pg) / ΟοΟ10 (μΜ); (±)5-8,12-iso-iPF2a-VI (pg) / (ω-3 / ω-6); (±)5-8,12-iso-iPF2a-VI (pg) / DHA (pg); (±)5-8,12-iso-iPF2a-VI (pg) / AA (pg); (±)5-8,12-iso-iPF2a-VI (pg) / % ω- 3; (±)5-8,12-iso-iPF2a-VI (pg) ^* BMI (Kg/m2) / AA (pg); (±)5-8,12-iso-iPF2a-VI (pg) ^* BMI (Kg/m2) / DHA (pg); (±)5-8,12-iso-iPF2a-VI (pg) / (β-carotene / %ω-3); (±)5- 8,12-iso-iPF2a-VI (pg) / (β-carotene / Ul ω-3 9); (±)5-8,12-iso-iPF2a-VI (pg) / (β- carotene / PI ω-3 10); (±)5-8,12-iso-iPF2a-VI (pg) / (Ul ω-3 / (CoQ10 / β-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) / (% ω-3 / (CoQ10 / ω-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) / (Ul ω-3/ (a-tocopherol / CoQ10 ^-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) / (% oj-3 / (a-tocopherol / CoQ10 ^-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) / (DHA / (a- tocopherol / CoQ10 ^-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) / (AA / (a-tocopherol / CoQ10 ^-carotene)); (±)5-8,12-iso-iPF2a-VI (pg) x BMI / (AA / (a-tocopherol / CoQ10 ^-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) ^* BMI / (DHA / (a-tocopherol / CoQ10 ^-carotene )); (±)5-8,12-iso-iPF2a-VI (pg) x BMI / (% ω-3 / (a-tocopherol / CoQ10 ^-carotene )); and (±)5-8,12-iso-iPF2a-VI (pg) x BMI / (Ul ω-3 / (a-tocopherol / CoQ10 Ζβ-carotene )), whereby when the ratio is above a control ratio is indicative that the pregnant woman is at risk of developing PE. [0076] Particularly, the ratio is calculated as: (±)5-8,12-iso-iPF2a-VI (pg) x BMI over (Ul ω-3 / (a-tocopherol /CoQ10 ^-carotene )), wherein ω-3 is omega-3 fatty acids = C18:3w3 + C18:4w3 + C20:3w3 + C20:4w3 + C20:5w3 + C22:5w3 + C22:6w3; Ul is Unsaturation index for omega-3 (Ul ω-3) = (%Monoenoic x 1 ) + (%Dienoic x 2) + (%Trienoic x 3) + (%Tetraenoic x 4) + (%Pentaenoic x 5) + (%Hexaenoic x 6) of ω-3 fatty acids, BMI is body mass index.

[0077] Alternatively, there is provided the assay as defined herein, wherein the blood-derived sample is selected from the group consisting of: blood, plasma, serum and blood cell membranes. Kit

[0078] The present invention also provides a diagnostic kit for performing the method of the present invention. The kit comprises reagents required to determine the level of the markers being measured. Suitable agents for assaying for the markers include enzyme-linked immunoassay reagents, RIA reagents and reagents for Western blotting.

[0079] A further aspect of the present invention relates to a kit for performing MS (in particular MS/MS) for quantifying class-VI isoprostanes in a subject's biological sample, this kit comprising: a standard for a 5-8,12 iso-iPF₂a -VI for calibration and validation; instructions for calibrating and validating the MS/MS, and instructions for measuring the class-VI isoprostane. Particularly, the standards are deuterated. More particularly the kit may also comprise standards for fatty acids ω-3 and/or ω-6 (such as arachidonic acid: AA) and/or phospholipids containing fatty acids ω-3 and/or ω-6.

Subject

[0080] Particularly, the subject is a pregnant woman. The sample can be taken at any time from about 10 weeks' gestation. Particularly, the sample can be taken at any time prior to the 24th week of pregnancy. More particularly, the sample is taken at between 12 and 20 weeks gestation. Sample

[0081] According to a particular aspect, the human subject biological sample may be any tissue that contains lipids. Particularly, isoprostanes can be detected and measured from samples such as urine, saliva, and blood-derived products such as, blood, plasma, serum and blood cell membranes.

[0082] More particularly, when a ratio over other lipids or fatty acids is desired for diagnostic purposes, a sample such as blood, plasma, serum and blood cell membranes is preferred.

[0083] The maternal sample can be any sample from which it is possible to measure the markers mentioned above. Particularly, the sample is selected from: blood, red or white blood cell membranes, plasma, serum, urine, or saliva. More particularly, the sample is taken from blood, plasma, serum or blood cell membranes. Most particularly, the sample is plasma or serum. More particularly, the markers are measured from blood cell membranes contained in the sample. Use as marker

[0084] More than twenty biochemical markers have been shown previously to be associated with established PE and there would be no logical prior reason for choosing a 5-8,12 iso-iPF_2a-VI in any prospective longitudinal study for assessment of use as predictive indicators. Moreover very few groups have evaluated any individual marker prospectively in the same women from whom samples were taken at intervals throughout their pregnancy. Importantly none has measured the different markers in the same women, unlike in the present application.

[0085] According to another aspect of the invention, there is provided that the use of a 5-8,12 iso-iPF₂a -VI and/or a ratio of a 5-8,12 iso-iPF₂a -VI over other parameters as a predictive marker(s) for pre-eclampsia in a pregnant woman, particularly, prior to 20^th week gestation, more particularly prior to the appearance of first symptoms.

Methodologies for measuring the markers

[0086] In accordance with another aspect of the present invention, there is provided a method for measuring blood isoprostane profile in a pregnant woman at risk of developing preeclampsia (PE), comprising the steps of: a) extracting lipids from the pregnant woman's biological sample; b) performing mass spectrometry on the extracted lipids to separate isoprostanes and measuring a 5-8,12 iso-iPF2a-VI ; c) comparing the level with a control level from a control population or individual representing the subject; d) reporting the comparison from step c) to the subject's treating physician; wherein when the level or ratio is at least about 15% higher than the control level or ratio, the physician may diagnose pre-eclampsia and, optionally take measures to monitor or treat the subject.

[0087] Alternatively, the method for measuring blood isoprostane profile in a pregnant woman at risk of developing preeclampsia (PE), further comprises the steps: b') assessing at least one parameter selected from: plasma volume, antioxidant level, body mass index and blood lipid profile in the sample; and b") establishing a ratio of isoprostane (±)5-8,12-iso-iPF2a-VI over at least one of a parameter selected from the group consisting of: plasma volume, body mass index, antioxidant levels, and blood lipid profile from the sample; c') comparing the ratio with a control ratio for a population or an individual representative of the pregnant woman; and d') determining if the comparing of step c') is higher than about 15% of the control ratio; and e') optionally reporting the determination from step f) to the subject's treating physician.

[0088] Particularly, the total fatty acid profile can be determined by liquid chromatography (LC), such as HPLC (high performance liquid chromatography), or HPLC-MS, or gas chromatography (GC) such as GC-FID (flame ionization detection) or GC-MS (mass spectrometry) or any other means well known in the art.

[0089] More particularly, the mass spectrometry technology used in step b) is: ion mobility MS that can be used before or after MS/MS fragmentation. [0090] Particularly, the level of a 5-8,12 iso-iPF_2a-VI can be assessed by one, two or more steps of mass spectrometry (MS-MS), particularly when preceded by liquid chromatography or by an ionization source such as for example: HPLC-MSMS, HPLC- MS-MS-MS; MALDI (Matrix-assisted laser desorption/ionization)-MS-MS, MALDI-MS- MS-MS, GC-MS-MS or ELISA or any other means well known in the art. [0091] More particularly, the levels of polyunsaturated fatty acids (PUFA) can be assessed by GC-FID (flame ionization detection), GC-MS or GC-MS-MS, HPLC- MS(/MS) LDTD-MS(/MS) or any other means well known in the art.

Immunoassay

[0092] Alternatively, the assay can take the form of an enzyme-linked immunoassay (ELISA) or a radio-immuno assay (RIA).

Therapeutic intervention

[0093] Particularly, the invention also comprises the additional step of taking measures to place the woman having an increased risk of PE under surveillance or tight monitoring for avoiding life threatening events for the foetus. Alternatively, the woman can be prescribed antioxidant therapy and monitored for further symptoms to develop or stabilize.

[0094] According to a further aspect, there is provided a method for treating a pregnant woman at risk of developing pre-eclampsia comprising: a) measuring (±)5- 8,12-isoprostane class-VI from a sample in accordance with the method above; b) determining that the ratio is at least above 15% of a control ratio; and c) administering an effective amount of a lipid selected from: AA, DHA, ω-3 fatty acids; a-tocopherol; v- tocopherol, vitamin E, CoQ10; and β-carotene to the pregnant woman to reach a ratio corresponding to a ratio of a control population; wherein the control population is a population of pregnant women characterized by normal blood pressure and absence of: obesity, diabetes, urine protein excretion and medical complications.

[0095] Particularly, the following lipid is administered to the pregnant woman in order to re-establish a control ratio: ω-3 fatty acids; a-tocopherol; γ-tocopherol, CoQ10; or β-carotene. Therapeutic target

[0096] Of course, an alternative aspect of the invention is to provide a marker useful for developing therapeutic strategies to avoid, prevent or treat PE.

[0097] The marker of the present invention may also be used in order to monitor the efficiency of a prophylactic treatment for preventing the development of PE, wherein a reduction in the risk of developing PE will be indicative of the efficacy of the prophylactic treatment.

[0098] The present invention offers many benefits. In addition to facilitating accurate targeting of interventions e.g. vitamin supplements or antioxidants, considerable saving on health care resources can be potentially gained due to stratification of antenatal care and reduced neonatal special care costs. In the research and development area, identification of high-risk patients will greatly facilitate future clinical trials. At present due to inadequate methods of prediction, large numbers of pregnant women unnecessarily receive interventions in clinical trials.

[0099] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

EXAMPLES EXAMPLE 1 - Materials and methods

Measurement of F_risoprostanes by HPLC-MS-MS

Materials

[00100] All F₂-isoprostanes and prostaglandin isomers, including 8-iso-15(R)- PGF_2a, Ent-8-iso-15(S)-PGF_2a, 8-iso-PGF_2a, Ent-8-iso-PGF_2a, 8-iso-PGF_2p, 1 1 -PGF_2a, 15(R)-PGF_2a, 5-trans-PGF_2a, PGF_2a, Ent-PGF_2a, PGF_2p, iPF_2a-IV, (±)5-iPF_2a-VI, (±)8,12-iso-iPF_2a-VI were purchased from Cayman Chemical (Ann Arbor, Ml, USA) as well as deuterated standards 8-iso-PGF_2a-d4, PGF_2a-d4, iPF_2a-IV-d4, iPF_2a-VI-d4, (±) 5-iPF_2a-VI-d1 1 , and (±)8,12-iso-iPF_2a-VI-d1 1 . Butylated hydroxytoluene (BHT) was bought from Sigma-Aldrich (Oakville, ON, Canada) and sodium chloride (ACS grade) was obtained from Laboratoire Mat (Quebec, QC, Canada). All other reagents and solvents were HPLC grade and were purchased from VWR International Inc. (Ville Mont-Royal, QC, Canada).

Preparation of solutions

[00101 ] A solution called internal standard containing 50 ng/mL of each deuterated analyte (8-iso-PGF_2a-d4, PGF_2a-d4, iPF_2a-IV-d4, iPF_2a-VI-d4, (±)5-iPF_2a-VI-d1 1 , and (±)8,12-iso-iPF_2a-VI-d1 1 ) was prepared in 0.01 % acetic acid. A stock solution containing 1 μg/mL of each compound (8-iso-15(R)-PGF_2a, 8-iso-PGF_2a, 15(R)-PGF_2a, 5-trans-PGF_2a, PGF_2a, iPF_2a-IV, (±)5-iPF_2a-VI and (±)8,12-iso-iPF_2a-VI) was also prepared in 0.01 % acetic acid. The previous solutions were used to prepare two sets of working solutions in which concentration ranged from 2 ng/mL to 80 ng/mL in 0.01 % acetic acid. First set of working solution was diluted to obtain standard curves for each analyte (10μΙ_ of working solution, 10 μΙ_ of internal standard, 80 μ Ι_ of water containing 10% (v/v) acetonitrile and 0.01 % (v/v) acetic acid). The second set of working solutions was diluted to obtain quality controls. Sample preparation

Erythrocyte cell membranes extraction [3]

[00102] Ten μ Ι_ of a BHT solution (1 % in ethanol) was added to 250 μ Ι_ of freshly thawed whole blood and the volume was completed to 1 ml with water. Samples were mixed, incubated for 5 min. at room temperature, and were centrifuged for 15 min. at 21 000 x g. The supernatant was discarded and 1 mL of a sodium chloride solution (0.9% (w/v) in water) was added. Samples were remixed and centrifuged for 12 minutes at 21 000 x g. The previous steps were done twice in order to wash correctly erythrocyte cell membranes. Finally, supernatant was discarded and 250 μ Ι_ of water was added to each tube. Aliquots were stored at -20^ until extraction of isoprostanes. Extraction of isoprostanes from erythrocyte cell membranes

[00103] Isoprostanes were extracted from erythrocyte cell membranes as described above from plasma but the totality of aliquots obtained after erythrocyte cell membranes extraction was used. No BHT solution was added in this case. Extraction of isoprostanes from plasma

[00104] Isopostanes were extracted from plasma using an adapted version of the method developed by Taylor [4]. Ten μ Ι_ of a BHT solution (1 % in ethanol) and 10 μΙ_ of the internal standard were added to 250 μΙ_ of freshly thawed plasma. Then, the samples were diluted with 250 μΙ_ of water and mixed with 500 μΙ_ of an hydrolysis solution (1 ml_ 50% (w/w) KOH, 1 ml_ water, 10 ml_ methanol). The resulting mixture was incubated at 37"C for 60 minutes. One hundred μ L of formic acid 0.05% (v/v) and 90 μΙ_ of hydrochloric acid 5N were added to each tube to stop the reaction. Samples were mixed and extracted twice with 1 .5 ml_ of hexane. The organic phase was discarded. The aqueous phase was then extracted three times with 1 .5 ml_ of 3:1 ethyl acetate: hexane. The organic phase was collected and combined in polypropylene conical tubes. Finally, extracts were evaporated to dryness under a stream of dry nitrogen and reconstituted with 100 μΙ_ of water containing 10% (v/v) acetonitrile and 0.01 % (v/v) acetic acid. Extraction of isoprostanes from whole blood

[00105] Isoprostanes were extracted from whole blood as described above for the plasma but 150 μΙ_ of blood was used instead. The samples were diluted to 350 μΙ_ with water. Only one extraction with hexane is performed though. After final reconstitution, the extract was filtered by a nanosep MF GHP .45μΜ at 13 000 RPM for 1 min. (Pall Life Science) before injection to the HPLC.

Chromatography

[00106] The chromatography was carried out using a Shimadzu Prominence system (Columbia, MD, USA). A Kinetex XB-C18 100 A column (100 x 3.0 mm, 2.6 μιτι) was used preceded by a 4.0 x 2.0 mm C18 SecurityGuard Cartridges. Both were from Phenomenex (Torrance, CA, USA). The column oven temperature was controlled at 3CC and the isoprostanes separation was perform ed using a gradient of three solvents at a flow rate of 0.45 mLJmin (see FIG. 1). Solvent A was composed of 0.01 % (v/v) acetic acid in water, solvent B consisted of 0.01 % (v/v) acetic acid in acetonitrile and solvent C was composed of 0.01 % (v/v) acetic acid in methanol. First, solvent B was held at 17% for 1 min, while solvent C was held at 33% followed by a linear gradient over 8.9 min to 13.5% B and 58.9% C. Then, a linear gradient over 0.5 min to 47.5% B and 47.5% C were programed. The latter conditions were maintained for 1 .6 min and were decreased to 17% B and 33% C in 0.1 min respectively. The final condition was held for 4.4 min to complete the 16.5 min run. The injection volume was 40 μΙ_ for samples, quality controls and the standard curve.

Mass Spectrometry

[00107] The HPLC was coupled to a 3200 QTRAP^® LC/MS/MS system from AB Sciex (Concord, ON, Canada) through a Turbo V™ ion source using the electrospray ionization probe according to the method described in Larose et al. [8]. The mass spectrometer was operated in negative mode. Curtain gas (CUR), collision gas (CAD), ion source gas 1 (GS1 ) and ion source gas 2 (GS2) were respectively set at 37, 7, 45 and 55. Ion spray voltage (IS) was set at -4100 V and source temperature was set at 700^. Class III F ₂-isoprostanes and their internal standard, 8-iso-PGF_2a-d4 and PGF_2a-d4 (class Nl-d4), were monitored in the multiple-reaction monitoring (MRM) mode using the transitions 353.3 / 193.2 and 357.3 / 197.2 respectively. Class IV F₂- isoprostanes and their internal standard, iPF_2a-IV-d4 (class IV-d4), were monitored using the transitions 353.3 / 127.0 and 357.0 / 127.0. Finally, class VI isoprostane and their internal standard, (±)5-iPF_2a-VI-d1 1 , and (±)8,12-iso-iPF_2a-VI-d1 1 (class Vl-d1 1 ), were analysed using the transitions 353.0 / 1 15.0 and 364.6 / 1 15.0 respectively. Table 1 summarizes analyte-specific mass spectrometry parameters for each transition. Quantification was performed using Analyst 1 .4.2^® Software.

Table 1. Multiple Reactions monitoring (MRM) transitions and analyte-specific MS parameters.

¹Declustering potential. ²Entrance potential. ¾ollision energy. "Collision cell entrance potential. ¾ollision cell exit potential.

Method validation

[00108] The lower limit of quantification (LLOQ) was defined as the concentration to which the S/N ratio was equal to 10 with precision below 20% and an accuracy of ± 20% of the nominal concentration. Determination of intra-day precision was done by analyzing a pool of plasma samples from three non-pregnant women (Innovative Research, Novi, Ml, USA) spiked with 10 μΙ_ of working solutions containing either 0 ng/mL, 7 ng/mL and 20 ng/mL of each analyte (n = 4 per concentration). This experiment was done on three consecutive days in order to evaluate inter-day precision (n = 12 per concentration). Concentration of each F₂-isoP was determined in a pooled plasma sample and accuracy was determined for the samples spiked with the 7 and 20 ng/mL solutions. The recovery was evaluated by comparing signal obtained for plasma spiked before extraction with 10 μΙ_ of solutions containing 7 ng/mL, 10 ng/mL and 20 ng/mL of each analyte with signal obtained for plasma spiked after extraction with the corresponding working solutions. Matrix effects were evaluated by post column infusion at 10 μΙ_Ληίη of a solution containing 100 ng/mL of each following molecules: 8-iso-PGF_2a, 8-iso-PGF_2a-d4, iPF_2a-IV, iPF_2a-IV-d4, 5-iPF_2a-VI , 5-iPF_2a-VI- d1 1 . During post column infusion, an extract of plasma was injected concomitantly using the described HPLC-MS/MS method above.

Fatty acid profile

[00109] The fatty acid composition of the plasma and erythrocyte membranes were performed according to the method previously described [ref. 5, 14]. The fatty acids from plasma were isolated according to a method previously described [6]. Briefly, a solution of chloroform:methanol (2:1 , by volume) was used to extract lipids from plasma. Then, phospholipids were separated by thin layer chromatography using a mix of isopropyl ether:acetic acid (96:4) as elutant and fatty acids were methylated following a trans esterification reaction using a mix of methanol:benzene (4:1 ) and acetyl chloride. Methylated fatty acids were finally analyzed by gas chromatography coupled with a flame ionization detector (GC-FID) as explained elsewhere [14, 15]. Ion mobility mass spectrometry

After the chromatographic separation of the isoprostanes as described above [ref. 9], the samples were introduced in a AB/SCIEX QTRAP® 6500 LC/MS/MS System equipped with a SelexlON™, ion mobility device. The parameters were optimized for each class VI F₂-isoprostane. A high concentration (3.0%) of 2-propanol was used as the differential mobility spectrometer (DMS) chemical modifier. The other operating parameters were set as follows: DMS temperature = 300Ό (high), DMS offset = 3.0 V, DMS resolution enhancement = low (22 psi) and separation voltage = 3750 V. According to those parameters, the optimal compensation voltage was -13.75 V for iPF_2a-VI and -10.62 V for both 5-iPF_2a -VI and (±)5-8,12-iso-iPF_2a -VI (FIG. 4B).

Relationship with antioxidant vitamins

[00110] Protein precipitation was carried out using 2 mL of methanol/ethanol (1/1 ) containing internal standards (4 ng of β-tocotrienol and 5 ng of ubiquinol-9) on 300 mL of plasma [ref. 9 and 10]. Vitamins were then extracted with 10 mL of hexane using the modified Menke's method [ref. 10]. After centrifugation, the hexane layer was removed, dried under a stream of nitrogen and resuspended in 700 mL of ethanol, then filtered before injection (10 mL) in the HPLC system. The HPLC mobile phase consisted of sonicated methanol/ethanol/isopropanol (88/24/10 v/v/v) containing 15 mM of lithium perchlorate at a flow of I mlJmin. The column was a Prontosil C18 (4.0 mm x 150 mm, 3 mm particle size) preceded by a Prontosil C18 guard cartridge (4.0 mm x 10 mm) (Bischoff Chromatography, Atlanta, GA). The coulometric electrochemical detector (Coulochem III, ESA, Bedford, MA) included a guard cell (Model 5020; coulometric electrode at -600 mV) and an analytical cell with two electrodes, the first one adjusted at -150 mV and the second at 600 mV. The current from the second electrode of the analytical cell was electronically recorded and data were integrated using the Beckman gold software (Fullerton, CA). Scales were adjusted at 2 mA for vitamin E, 100 nA for ubiquinol-10 and β-carotene, and 50 nA for ubiquinone-10. The amounts of the lipophilic antioxidants were calculated from the ratios of the peak areas of these components to the corresponding internal standard. The γ-tocotrienol was used as an internal standard for γ-tocopherol and a-tocopherol. The ubiquinol-9 was used as an internal standard for β-carotene, ubiquinol-10, and ubiquinone-10 [ref. 9].

TABLE 2. Effect of normalization with lipids and antioxidants on the F₂-isoprostanes levels in the first and second trimester between controls and women that will later develop preeclampsia during the course of their pregnancies.

Visit #1 (12-18 weeks)¹

Isoprostanes / Normotensive Preeclampsia

controls

Normalization factors (n=33) (Area, ROC

(n=25) curve)

(iPFzi-VI + 5 iPFzi-VI) (pg) / plasma volume (ml) 0.0496*

154 [124, 175] 172 [137, 209]

PRIOR ART from WO 2013/170369 (0.6515)

0.0076*

609 [481, 713] 764 [594, 998]

(±)5-8, 1 2-iso-iPF_2a-VI (pg) / plasma volume (ml) (0.7042)

0.0080*

757 [597, 897] 942 [730, 1203]

Sum of Class VI (pg) / plasma volume (ml) (0.7030)

0.0068*

15222 [9968, 20172 [13217,

(±)5-8, 12-iso-iPF_2a-VI (pg) x BMI ⁴ (Kg/m²) / plasma volume (ml) 16972] 30086]

(0.7133)

0.0002*

17.4 [14.2, 20.8] 24.5 [18.6, 35.5]

(±)5-8, 1 2-iso-iPF_2a-VI (pg) / -tocopherol (μΜ) (0.7794)

0.0331*

253 [192, 331] 330 [232, 424]

(±)5-8,1 2-iso-iPF_2a-VI (pg) / γ-tocopherol (μΜ) (0.6642)

Visit #1 (12-18 weeks)¹

Isoprostanes / Normotensive Preeclampsia

Normalization factors controls (n=33) (Area, ROC

(n=25) curve)

0.0344*

34.5 [27.9, 58.8] 61.8 [32.8, 126]

(±)5-8, 12-iso-iPF_2a-VI (pg) / ( -tocopherol / γ-tocopherol) (0.6630)

0.0001*

16.5 [12.9, 19.5] 21.5 [17.6, 31.8]

(±)5-8,12-iso-iPF_2a-VI (pg) / vitamin E (μΜ) ⁵ (0.7891)

<0.0001**

660 [341, 1065] 2406 [1576, 5146]

(±)5-8, 12-iso-iPF_2a-VI (pg) / β-carotene (μΜ) (0.8642)

0.0027*

492 [315, 597] 688 [573, 918]

(±)5-8,12-iso-iPF_2a-VI (pg) / CoQ₁₀ ^M) (0.7285)

3823 [2835, 0.0024*

5927 [3595, 7369]

(±)5-8, 12-iso-iPF_2a-VI (pg) / (co-3 / co-6) ⁶'⁷ 4776] (0.7309)

0.0012*

11.3 [8.1, 13.9] 16.9 [11.0, 22.4]

(±)5-8, 12-iso-iPF_2a-VI (pg) / DHA ^g) (0.7455)

0.0011*

3.9 [3.4, 5.0] 5.1 [4.5, 6.6]

(±)5-8, 12-iso-iPF_2a-VI (pg) / AA fag) (0.7467)

Visit #1 (12-18 weeks)¹

Isoprostanes / Normotensive Preeclampsia

controls

Normalization factors (n=33) (Area, ROC

(n=25) curve)

0.0013*

107 [83, 133] 165 [101, 201]

(±)5-8,1 2-iso-iPF_2a-VI (pg) / % co-3 ⁸ (0.7442)

0.0004

64761 [39591, 116393 [69222,

(±)5-8, 12-iso-iPF_2a-VI (pg) x BMI (Kg/m²) / AA ^g) 86443] 180046]

(0.7731)

0.0006*

158762 [111482, 332591 [167942,

(±)5-8, 1 2-iso-iPF_2a-VI (pg) x BMI (Kg/m²) / DHA ^g) 246323] 638705]

(0.7663)

<0.0001**

3509 [1917, 11 558 [8225, 25

(±)5-8,1 2-iso-iPF_2a-VI (pg) / (β-carotene / % co-3) 6155] 966]

(0.8461)

<0.0001**

19 643 [10 683, 63 305 [45 778,

(±)5-8, 1 2-iso-iPF_2a-VI (pg) / (β-carotene / UI co-3 ⁹) 34 574] 143 996]

(0.8424)

<0.0001**

25 251 [13 701, 80 379 [58 654,

(±)5-8, 12-iso-iPF_2a-VI (pg) / (β-carotene / PI co-3 ¹⁰) 44 526] 184 130]

(0.8388)

<0.0001**

24.0 [16.4, 46.9] 110 [49.4, 231]

(±)5-8,1 2-iso-iPF_2a-VI (pg) / (UI co-3 / (CoQlO / β-carotene )) (0.8641)

Visit #1 (12-18 weeks)¹

Isoprostanes / Normotensive Preeclampsia

Normalization factors controls (n=33) (Area, ROC

(n=25) curve)

<0.0001**

130 [92.2, 265] 596 [273, 1291]

(±)5-8, 12-iso-iPF_2a-VI (pg) / (% co-3 / (CoQlO / β-carotene )) (0.8473)

<0.0001**

(±)5-8, 12-iso-iPF_2a-VI (pg) / (UI co-3/ (a-tocopherol / CoQlO /β- 673 [244, 1358] 2946 [983, 5013]

carotene )) (0.8521)

3807 [1359, 16 398 [5406, 28 <0.0001**

(±)5-8,12-iso-iPF_2a-VI (pg) / (% co-3 / (a-tocopherol / CoQlO /β- 7628] 099]

carotene )) (0.8558)

<0.0001**

(±)5-8, 12-iso-iPF_2a-VI (pg) / (DHA / (a-tocopherol / CoQlO /β- 319 [142, 863] 1589 [584, 3554]

carotene )) (0.8412)

<0.0001**

(±)5-8, 12-iso-iPF_2a-VI (pg) / (AA / (a-tocopherol / CoQlO /β- 125 [69, 284] 550 [195, 1264]

carotene )) (0.8303)

3075 [1463, 13801 [6944, <0.0001**

(±)5-8, 12-iso-iPF_2a-VI (pg) x BMI / (AA / (a-tocopherol / CoQlO 5389] 32824]

^-carotene )) (0.8492)

8768 [3129, 45959 [18107, <0.0001**

(±)5-8,12-iso-iPF_2a-VI (pg) x BMI / (DHA / (a-tocopherol 15403] 84089]

/ CoQlO ^-carotene )) (0.8573)

Visit #1 (12-18 weeks)¹

Isoprostanes / Normotensive Preeclampsia

Normalization factors controls (n=33) (Area, ROC

(n=25) curve)

82621 [30784, 401259 [173111, <0.0001**

(±)5-8, 12-iso-iPF_2a-VI (pg) x BMI / (% co-3 / ( -tocopherol 188696] 707942]

/ CoQIO /p-carotene )) (0.8601)

14937 [5524, 74493 [30717, <0.0001**

(±)5-8, 12-iso-iPF_2a-VI (pg) x BMI / (UI co-3 / (a-tocopherol 33887] 127269]

/ CoQIO /p-carotene )) (0.8628)

*P < 0.05

**P <0.0001

^Values are medians and quartiles [Q1 , Q3]

² Mann- Whitney test

ROC = receiver operating characteristic

⁴ pre-pregnancy body mass index (BMI)

⁵ Vitamin E = D-tocopherol + D-tocopherol

⁶ Omega-3 fatty acids = C18:3co3 + C18:4co3 + C20:3co3 + C20:4co3 + C20:5co3 + C22:5co3 + C22:6co3

⁷ Omega-6 fatty acids = C18:lco6 + C18:2co6 + C18:3co6 + C20:2co6 + C20:3co6 + C20:4co6 + C22:2co6 + C22:4co6 + C22:5co6

⁸ ω-3 % = all co-3 described in [⁶ ] / total of all fatty acids described x 100 in ref 8.

⁹ Peroxidation index for omega-3 (PI co-3) = (%Trienoic x 2) + (%Tetraenoic x 4) + (%Pentaenoic x 6) + (%Hexaenoic x 8) of fatty acids described above in [⁶ ]

¹⁰ Unsaturation index for omega-3 (UI co-3) = (%Monoenoic x 1 ) + (%Dienoic x 2) + (%Trienoic x 3) + (%Tetraenoic x 4) + (%Pentaenoic x 5) + (%Hexaenoic x 6) of fatty acids described above in [⁶ ]

EXAMPLE 2 - Results

[00111 ] We have improved the separation of isoprostanes of class VI using the newly developed ion mobility in comparison to our previously published method [ref. 7]. This additional step brings a new dimension of separation to the traditional HPLC- MS/MS (FIG. 4A). This technology also improves the limit of detection, the signal to noise ratio and enhances the concentration dynamic range and is particularly suitable for lipid isomer separation [ref. 1 1 ].

[00112] The newly discovered (±)5-8,12-iso-iPF_2a-VI biomarker is clearly superior to the combined measure of iPF_2a-VI + 5 iPF_2a-VI reported earlier in WO 2013-170369 and [ref. 15] for preeclampsia prediction as shown in Table 2. Indeed, the new marker is 25.5% higher in preeclampsia than controls (P = 0.0076) while the previous marker is 1 1 .7% higher than uneventful pregnancies per unit of plasma volume (P= 0.0496). Table 2 also shows different ways to normalize isoprostanes plasma data according to pre-pregnancy body mass index (BMI), antioxidant vitamins and fatty acids content, all factors suspected to influence the level of oxidative stress linked to isoprostanes production in preeclampsia [ref. 12].

[00113] These results comprise a subset of women reported in WO 2013/170369 from which antioxidant plasma vitamins data was available such as β-carotene, vitamin E and coenzyme Q₁₀ (CoQi₀) at the first (12-18 weeks) routine visit for the pregnancy follow-up. Drastic improvement of the preeclampsia (PE) prediction was observed with pre-pregnancy BMI, a-tocopherol, β-carotene, ω-3 fatty acids as normalizing factors in ratio (ROC curve area = 0.8628 (FIG. 5)). Of note, a low β-carotene level during pregnancy is predictive of the later occurrence of PE in our cohort (ROC curve area of 0.8382). The plasma levels of β-carotene from 25 normotensive controls (1 .07 ± 0.13; mean ± SEM) was 2.5-fold-higher than PE pregnancies (0.42 ± 0.08 n=33, P <0.0001 Mann Whitney test) at 12-18 weeks of pregnancies. Indeed, β-carotene levels were also reported lower in severe cases of PE or in PE complicated by diabetes [ref 13].

[00114] Moreover, a deficient omega-3 level is also a feature of preeclamptic group when compared to controls as reported by us (p=0.03) [ref 15]. These two respective latter deficiencies are still controversial and may be specific to our cohort though; this is why we propose to control for these levels of β-carotene and omega-3 fatty acids together with pre-pregnancy BMI, vitamin E isomer α-tocopherol and coenzyme Q10, a vitamin-like antioxidant. In taking into account all these cofactors, very good prediction of preeclampsia can be reached (FIG. 5).

Example 3 - Lactone formation of (±)5-8, 12-iso-iPF_2a -VI

[00115] The biomarker, (±)5-8,12-iso-iPF2a -VI, can be transformed into 5, 8,12- iso-iPF2a-VI-1 ,5-lactone under any acidic conditions (FIG. 6). Therefore, the biomarker can alternatively be quantified under acidic condition using the transition 337.3 → 319.3 m/z in the positive mode of the mass spectrometer. The same chromatographic methodology can be used for both forms (isoprostane and lactone).

[00116] Moreover, transformation into lactones can also be used to separate all class-VI isomers from other F2-isoPs through chromatography or other means. The alkaline condition would then regenerate the biomarker in its initial form, (±)5-8,12-iso- iPF2a -VI.

Example 4 - Derivatization of the (±)5-8, 12-iso-iPF₂a -VI

[00117] It is known that F2-isoPs can be derivatized to increase sensitivity, for example, using pentafluorobenzyl ester (FIG. 7) [ref. 18], or other derivatives such as: 4-diazomethylpyridine [ref. 19]; or 2-diethylaminoethyl chloride, 2-diethylaminoethyl bromide [ref. 20]; or trimethylanilinium hydroxide (TMAH) [ref. 21 ]; or 2,4- bis(diethylamino)-6-hydrazino-1 ,3,5-triazine [ref. 22], as non-limiting examples.

Example 5- Detection by Laser Diode Thermal Desorption (LDTD)

[00118] The LDTD replaces the HPLC or GC and is directly coupled to the mass spectrometer with or without ion mobility. One of the advantages of LDTD over HPLC is less ionic suppression that could lead to better sensitivity. The LDTD/APCI source parameters were ramped and optimized for 6 μΙ of extracted samples in 0.1 mg/mL EDTA, 0.75% NH₄OH, 37.4% methanol, 12.5% water per well using a LazWell HDE Teflon coated plate. The plate is dried before analysis. Corona discharge needle voltage, vaporizer temperature and carriers gas airflow rates are also optimized. The laser pattern used was 6-45-2. The LazSoft 4.0 (Phytronix) controlled the LDTD/APCI source within Analyst 1 .6.2 (Sciex).

[00119] We were also able to alternatively measure the (±)5-8,12-iso-iPF2a -VI in the low pg range using LDTD as shown in the lower panel of FIG. 8. The quantification is linear over several logs (FIG. 8, top panel). The extraction of the samples for LDTD was carried out using solid supported liquid extraction (SLE) and solid phase extraction (SPE). These methods can be used successively in dirty matrixes like tissues. Briefly, 0.2 ml of plasma in 100 mM pH=3 acetate buffer containing deuterated standard is loaded on a SLE cartridge (Phenomenex Novum SLE 3 cc). The (±)5-8,12-iso-iPF2a - VI is eluted with 3 times 0.6 ml Methyl tert-butyl ether (MTBE): Hexane (8:2). The eluate is evaporated to dryness under a stream of nitrogen and reconstituted with 0.1 ml of 13.5% acetonitrile, 31 .5% methanol and water containing 0.01 of acetic acid. For the SPE, 1 ml of plasma spiked with internal standard is diluted in 2 ml of acetate buffer (100 mM, pH=3) and loaded on preconditioned SPE cartridge (Phenomenex Strata-X 100 mg, 6 ml) with methanol. The SPE cartridge is washed twice with 2 ml of hexane. The (±)5-8,12-iso-iPF2a -VI is eluted 2 x 1 ml of ethyl acetate. The eluate is evaporated to dryness and reconstituted with 0.1 ml of 13.5% acetonitrile, 31 .5% methanol and water containing 0.01 % of acetic acid.

Threshold of detection for pre-eclampsia (PE)

[00120] An increase as low as 15% (or more using the median) of class VI isomers like of (±)5-8,12-iso-iPF_2a-VI normalized to the n-3/n-6 ratio in comparison to the control group, is indicative of PE (FIG. 9). The typical variations are within 20 to 45% above controls. An increase of class VI concomitant to an increase of class III or IV isomers between 20% to 200% compared to controls could be indicative of other unrelated conditions.

[00121 ] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

[00122] All patents, patent applications and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent, patent application or publication was specifically and individually indicated to be incorporated by reference. References

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2. Xu, H., Perez-Cuevas, R., Xiong, X., Reyes, H ., Roy, C, Julien, P., Smith, G., von Dadelszen, P., Leduc, L, Audibert, F., Moutquin, J. M., Piedboeuf, B., Shatenstein, B., Parra- Cabrera, S., Choquette, P., Winsor, S., Wood, S., Benjamin, A., Walker, M., Helewa, M., Dube, J., Tawagi, G., Seaward, G., Ohlsson, A., Magee, L. A., Olatunbosun, F., Gratton, R., Shear, R., Demianczuk, N., Collet, J. P., Wei, S., Fraser, W. D., and group, I. s. (2010) An international trial of antioxidants in the prevention of preeclampsia (INTAPP). Am J Obstet Gynecol 202, 239 e231 -239 e210..

3. Counil, E., Julien, P., Lamarche, B., Chateau-Degat, M. L, Ferland, A., and Dewailly, E. (2009) Association between trans-fatty acids in erythrocytes and pro-atherogenic lipid profiles among Canadian Inuit of Nunavik: possible influences of sex and age. Br J Nutr 102, 766-776.

4. Taylor, A. W., Bruno, R. S., Frei, B., and Traber, M. G. (2006) Benefits of prolonged gradient separation for high-performance liquid chromatography-tandem mass spectrometry quantitation of plasma total 15-series F-isoprostanes. Anal Biochem 350, 41 -51 .

5. Roland, L, Gagne, A., Belanger, M. C, Boutet, M., Berthiaume, L, Fraser, W. D., Julien, P., and Bilodeau, J. F. (2010) Existence of compensatory defense mechanisms against oxidative stress and hypertension in preeclampsia. Hypertens Pregnancy 2Q, 21 -37.

6. Nigam, A., Frasure-Smith, N., Lesperance, F., and Julien, P. (2009) Relationship between n-3 and n-6 plasma fatty acid levels and insulin resistance in coronary patients with and without metabolic syndrome. Nutr Metab Cardiovasc Dis 19, 264-270.

7. Larose, J., Julien, P. , and Bilodeau, J. F. (2013) Analysis of F2-isoprostanes in plasma of pregnant women by H PLC-MS/MS using a column packed with core-shell particles. J Lipid Res 54, 1505-151 1 .

8. Finckh, B., Kontush, A., Commentz, J., Hubner, C, Burdelski, M., and Kohlschutter, A. (1995) Monitoring of ubiquinol-10, ubiquinone-10, carotenoids, and tocopherols in neonatal plasma microsamples using high-performance liquid chromatography with coulometric electrochemical detection. Anal Biochem 232, 210-216.

9. Belanger, M. C, Mirault, M. E., Dewailly, E., Berthiaume, L., and Julien, P. (2008) Environmental contaminants and redox status of coenzyme Q10 and vitamin E in Inuit from Nunavik. Metabolism 57, 927-933..

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15. Bilodeau, J. F., Qin Wei, S., Larose, J., Greffard, K., Moisan, V., Audibert, F., Fraser, W. D., and Julien, P. (2015) Plasma F2-isoprostane class VI isomers at 12-18 weeks of pregnancy are associated with later occurrence of preeclampsia. Free Radio Biol Med 85, 282- 287.

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Claims

1. A method for measuring (±)5-8, 12-isoprostane class-VI in a human subject, comprising the steps of:

a) obtaining a biological sample from said patient;

b) extracting lipids from said sample;

c) optionally, separating said (±)5-8, 12-isoprostane class-VI [(±)5-8, 12-iso- iPF_2a-VI] from said extracted lipids; and

d) measuring a level of isoprostane (±)5-8, 12-iso-iPF_2a-VI.

2. The method according to claim 1 , wherein said separating is carried out by gas, liquid or ion mobility chromatography.

The method according to claim 2, wherein said separating is carried out

HPLC.

4. The method according to claim 1 , 2 or 3, wherein said measuring is carried out by mass spectrometry (MS). 5. The method according to claim 1 , wherein said separating and measuring steps are carried out by MS coupled to an ion-mobility device.

6. The method of claim 5, wherein said separating and measuring is carried out with Laser Diode Thermal Desorption coupled to mass spectrometry (LDTD-MS).

7. The method according to any one of claims 1 to 6, further comprising a step of: c') treating (±)5-8, 12-iso-iPF_2a -VI under acidic conditions to produce 5, 8, 12- iso-iPF2a-VI-1 ,5-lactone; and

d) measuring level of said lactone.

8. The method according to any one of claims 1 to 6, further comprising a step of: c') derivatizing (±)5-8, 12-iso-iPF_2a -VI into a derivative thereof; and

d) measuring a level of isoprostane (±)5-8, 12-iso-iPF_2a-VI derivative thereof.

9. The method according to claim 8, wherein said derivative is selected from the group consisting of: pentafluorobenzyl ester, 4-diazomethylpyridine, 2-diethyl aminoethyl chloride, 2-diethylaminoethyl bromide, trimethylanilinium hydroxide, and 2,4-bis(diethylamino)-6-hydrazino-1 ,3,5-triazine.

10. The method according to any one of claims 1 to 9, wherein said subject is a pregnant woman, the method further comprising:

comparing said level from step d) with a control level from a control population representative of said pregnant woman; and reporting said comparison to said pregnant woman's treating physician;

whereby when said level is at least about 15% higher than said control level, said physician may diagnose pre-eclampsia and, optionally take measures to monitor or treat said pregnant woman.

11. The method according to any one of claims 1 to 9, wherein said subject is a pregnant woman, the method further optionally comprising:

e) measuring from said sample a parameter selected from: plasma volume, antioxidant level, body mass index and blood lipid profile;

f) establishing a ratio of (±)5-8, 12-iso-iPF_2a-VI over one of said parameter from step e) for said subject;

g) comparing said ratio from step f) with a control ratio from a control population representative of said pregnant woman;

h) reporting said comparison from step g) to said pregnant woman's treating physician;

whereby when said level or ratio is at least about 15% higher than said control level or control ratio, said physician may diagnose pre-eclampsia and, optionally take measures to monitor or treat said pregnant woman.

12. The method according to claim 11 , wherein said ratio is selected from the group consisting of:

(±)5-8, 12- ^■iso- iPF_2a- ^■VI (pg) / plasma volume (ml);

(±)5-8, 12- ^■iso- iPF_2a- ^■VI (pg) x BMI (Kg/m²) / plasma volume (ml);

(±)5-8, 12- ^■iso- iPF_2a- ^■VI (pg) / a-tocopherol (μΜ);

(±)5-8, 12- ^■iso- iPF_2a- ^■VI (pg) / γ-tocopherol (μΜ);

(±)5-8, 12- ^■iso- iPF_2a- ^■VI (pg) / a-tocopherol / γ-tocopherol);

(±)5-8, 12- ^■iso- iPF_2a- ^■VI (pg) / vitamin E (μΜ);

(±)5-8, 12- ^■iso- iPF_2a- ^■VI (pg) / β-carotene (μΜ); (±)5-8, 12-iso-iPF_2a-VI (pg) / ΟοΟ₁₀ (μΜ);

(±)5-8, 12-iso-iPF_2a-VI (pg) / (ω-3 / ω-6);

(±)5-8, 12-iso-iPF_2a-VI (pg) / DHA ( g);

(±)5-8, 12-iso-iPF_2a-VI (pg) / AA ( g);

(±)5-8, 12-iso-iPF_2a-VI (pg) / % ω-3;

(±)5-8, 12-iso-iPF_2a-VI (pg) x BMI (Kg/m²) / AA ^g);

(±)5-8, 12-iso-iPF_2a-VI (pg) x BMI (Kg/m²) / DHA ^g);

(±)5-8, 12-iso-iPF_2a-VI (pg) / (β-carotene / %ω-3);

(±)5-8, 12-iso-iPF_2a-VI (pg) / (β-carotene / Ul ω-3);

(±)5-8, 12-iso-iPF_2a-VI (pg) / (β-carotene / PI ω-3);

(±)5-8, 12-iso-iPF_2a-VI (pg) / (Ul ω-3 / (CoQ10 / β-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) / (% ω-3 / (CoQ10 / ω-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) / (Ul ω-3/ (a-tocopherol / CoQ10 ^-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) / (% ω-3 / (a-tocopherol / CoQ10 ^-carotene )); (±)5-8, 12-iso-iPF_2a-VI (pg) / (DHA / (a-tocopherol / CoQ10 ^-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) / (AA / (a-tocopherol / CoQ10 ^-carotene));

(±)5-8, 12-iso-iPF_2a-VI (pg) x BMI / (AA / (a-tocopherol / CoQ10 ^-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) x BMI / (DHA / (a-tocopherol / CoQ10 ^-carotene )); (±)5-8, 12-iso-iPF_2a-VI (pg) x BMI / (% ω-3 / (a-tocopherol / CoQ10 ^-carotene )); and

(±)5-8, 12-iso-iPF_2a-VI (pg) x BMI / (Ul ω-3 / (a-tocopherol / CoQ10 ^-carotene )); wherein BMI is body mass index, Ul ω-3 is an unsaturation index for omega-3 fatty acids corresponding to the formula: (%Monoenoic x 1) + (%Dienoic x 2) + (%Trienoic x 3) + (%Tetraenoic x 4) + (%Pentaenoic x 5) + (%Hexaenoic x 6); said omega-3 fatty acids being: C18:3w3 and C18:4w3 and C20:3w3 and C20:4w3 and C20:5w3 and C22:5w3 and C22:6w3; and PI ω-3 is a peroxidation index for omega-3 fatty acids corresponding to: (%Trienoic x 2) + (%Tetraenoic x 4) + (%Pentaenoic x 6) + (%Hexaenoic x 8);

whereby when said ratio is at least about 15% above a control ratio, then said ratio is indicative that said pregnant woman is at risk of developing PE.

13. The method according to claim 12, wherein said ratio is a ratio selected from the group consisting of:

(±)5-8, 12-iso-iPF_2a-VI (pg) / β-carotene (μΜ); (±)5-8, 12-iso-iPF_2a-VI (pg) / (β-carotene / % ω-3);

(±)5-8, 12-iso-iPF_2a-VI (pg) / (β-carotene / Ul ω-3);

(±)5-8, 12-iso-iPF_2a-VI (pg) / (β-carotene / PI ω-3);

(±)5-8, 12-iso-iPF_2a-VI (pg) / (Ul ω-3 / (CoQ10 / β-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) / (% ω-3 / (CoQ10 / β-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) / (Ul ω-3/ (a-tocopherol /CoQ10 /p-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) / (% ω-3 / (a-tocopherol /CoQ10 ^-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) / (DHA / (a-tocopherol /CoQ10 ^-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) / (AA / (a-tocopherol /CoQ10 ^-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) x BMI / (AA / (a-tocopherol /CoQ10 ^-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) x BMI / (DHA / (a-tocopherol /CoQ10 ^-carotene )); (±)5-8, 12-iso-iPF_2a-VI (pg) x BMI / (% ω-3 / (a-tocopherol /CoQ10 ^-carotene )); and

(±)5-8, 12-iso-iPF_2a-VI (pg) x BMI / (Ul ω-3 / (a-tocopherol /CoQ10 ^-carotene )). 14. The method according to claim 13, wherein said ratio is calculated as:

(±)5-8.12-iso-iPF2a-VI (pg) x BMI

(Ul ω-3 / (a-tocopherol /CoQ10 ^-carotene )).

15. The method according to any one of claims 1 to 10, wherein said biological sample is selected from the group consisting of: urine, saliva, blood, plasma, serum and blood cell membranes.

16. The method according to any one of claims 11 to 14, wherein said biological sample is selected from the group consisting of: blood, plasma, serum and blood cell membranes.

17. The method according to any one of claims 10 to 14, wherein said control population is a population of pregnant women characterized by a normal blood pressure and absence of: obesity, diabetes, urine protein excretion and medical complications.

18. The method according to any one of claims 10 to 17, wherein said level or said ratio is increased by at least about 20%, 25%, 50%, 100%, 200%, 300% or even higher than 400%.

19. A method for predicting the appearance of preeclampsia (PE) in a pregnant woman comprising the steps of:

a) obtaining a sample from said pregnant woman;

b) measuring a level of isoprostane (±)5-8, 12-iso-iPF_2a-VI in said sample;

c) comparing said level with a control level for a population or an individual representative of said pregnant woman; and

d) determining if said comparing of step c) is higher than about 15% of said control level; and

e) optionally reporting said determination from step f) to said subject's treating physician.

20. The method according to claim 19, further comprising step:

b') assessing at least one parameter selected from: plasma volume, body mass index and blood lipid profile in said sample; and

b") establishing a ratio of isoprostane (±)5-8, 12-iso-iPF_2a-VI over at least one of a parameter selected from the group consisting of: plasma volume, body mass index and blood lipid profile from said sample;

c') comparing said ratio with a control ratio for a population or an individual representative of said pregnant woman; and

d') determining if said comparing of step c') is higher than about 10% of said control ratio; and

e') optionally reporting said determination from step f) to said subject's treating physician.

21. The method according to claim 19 or 20, wherein said control level or ratio is established with a control population or individual, wherein said control population is a population of pregnant women characterized by a normal blood pressure and absence of: obesity, diabetes, urine protein excretion and medical complications.

22. The method according to any one of claims 19 to 21 , wherein said level or said ratio is increased by at least about 20%, 25%, 50%, 100%, 20%, 300% or even greater than 400%. 23. The method according to any one of claims 20 to 22, wherein said ratio is selected from a group consisting of: ±)5-8, ' 12- iso- PF_2a ^■VI (pg) / plasma volume (ml);

±)5-8, 12- iso- PF_2a ^■VI (pg) x BMI (Kg/m²) / plasma volume (ml);

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / a-tocopherol (μΜ);

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / γ-tocopherol (μΜ);

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / a-tocopherol / γ-tocopherol);

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / vitamin E (μΜ);

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / β-carotene (μΜ);

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / ΟοΟ₁₀ (μΜ);

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / (ω-3 / ω-6);

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / DHA (_Mg);

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / ΑΑ ^g);

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / % ω-3;

±)5-8, ' 12- iso- PF_2a ^■VI (pg) χ ΒΜΙ (Kg/m²) / AA ^g);

±)5-8, ' 12- iso- PF_2a ^■VI (pg) χ BMI (Kg/m²) / DHA ^g);

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / (β-carotene / %ω-3);

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / (β-carotene / Ul ω-3) ;

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / (β-carotene / PI ω-3);

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / (Ul ω-3 / (CoQ10 / β-carotene ));

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / (% ω-3 / (CoQ10 / ω-carotene ));

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / (Ul ω-3/ (α-tocopherol / CoQ10 ^-carotene ));

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / (% oj-3 / (α-tocopherol / CoQ10 ^-carotene ));

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / (DHA / (α-tocopherol / CoQ10 ^-carotene ));

±)5-8, ' 12- iso- PF_2a ^■VI (pg) / (AA / (α-tocopherol / CoQ10 ^-carotene));

±)5-8, ' 12- iso- PF_2a ^■VI (pg) x BMI / (AA / (α-tocopherol / CoQ10 ^-carotene ));

±)5-8, ' 12- iso- PF_2a ^■VI (pg) x BMI / (DHA / (α-tocopherol / CoQ10 ^-carotene ));

±)5-8, ' 12- iso- PF_2a ^■VI (pg) x BMI / (% OJ-3 / (α-tocopherol / CoQ10 ^-carotene )); and

±)5-8, ' 12- iso- PF_2a ^■VI (pg) x BMI / (Ul OJ-3 / (α-tocopherol / CoQ10 ^-carotene )) wherein BMI is body mass index, Ul OJ-3 is an unsaturation index for omega-3 fatty acids corresponding to the formula: (%Monoenoic x 1 ) + (%Dienoic x 2) + (%Trienoic x 3) + (%Tetraenoic x 4) + (%Pentaenoic x 5) + (%Hexaenoic x 6); said omega-3 fatty acids being: C18:3oo3 and C18:4oo3 and C20:3oo3 and C20:4oo3 and C20:5oo3 and C22:5OJ3 and C22:6oo3; and PI OJ-3 is a peroxidation index for omega-3 corresponding to (%Trienoic x 2) + (%Tetraenoic x 4) + (%Pentaenoic x 6) + (%Hexaenoic x 8) of said fatty acids; whereby when said ratio is above a control ratio is indicative that said pregnant woman is at risk of developing PE.

24. The method according to claim 23, wherein said ratio is a ratio selected from the group consist ng of:

(±)5-8, 12- so-iPF_2a-VI (pg) / β-carotene (μΜ);

(±)5-8, 12- so-iPF_2a-VI (pg) / (β-carotene / % ω-3);

(±)5-8, 12- so-iPF_2a-VI (pg) / (β-carotene / Ul ω-3);

(±)5-8, 12- so-iPF_2a-VI (pg) / (β-carotene / PI ω-3);

(±)5-8, 12- so-iPF_2a-VI (pg) / (Ul ω-3 / (CoQ10 / β-carotene ));

(±)5-8, 12- so-iPF_2a-VI (pg) / (% ω-3 / (CoQ10 / β-carotene ));

(±)5-8, 12- so-iPF_2a-VI (pg) / (Ul ω-3/ (a-tocopherol /CoQ10 ^-carotene ));

(±)5-8, 12- so-iPF_2a-VI (pg) / (% ω-3 / (a-tocopherol /CoQ10 ^-carotene ));

(±)5-8, 12- so-iPF_2a-VI (pg) / (DHA / (a-tocopherol /CoQ10 ^-carotene ));

(±)5-8, 12- so-iPF_2a-VI (pg) / (AA / (a-tocopherol /CoQ10 ^-carotene ));

(±)5-8, 12- so-iPF_2a-VI (pg) x BMI / (AA / (a-tocopherol /CoQ10 ^-carotene )); (±)5-8, 12- so-iPF_2a-VI (pg) x BMI / (DHA / (a-tocopherol /CoQ10 ^-carotene )); (±)5-8, 12-iso-iPF_2a-VI (pg) x BMI / (% ω-3 / (a-tocopherol /CoQ10 ^-carotene )); and

(±)5-8, 12-iso-iPF_2a-VI (pg) x BMI / (Ul ω-3 / (a-tocopherol /CoQ10 ^-carotene )).

25. The method according to claim 24, wherein said ratio is calculated as:

(±)5-8.12-iso-iPF2a-VI (pg) x BMI

(Ul ω-3 / (a-tocopherol /CoQ10 ^-carotene )).

26. The method according to claim 19, wherein said biological sample is selected from the group consisting of: urine, saliva, blood, plasma, serum and blood cell membranes.

27. The method according to any one of claims 20 to 25, wherein said biological sample is selected from the group consisting of: blood, plasma, serum and blood cell membranes.

28. A method for treating a pregnant woman at risk of developing pre-eclampsia comprising: a) measuring (±)5-8, 12-isoprostane class-VI from a sample in accordance with claim 10;

b) determining that said ratio is at least above 15% of a control ratio; and c) administering an effective amount of a lipid selected from: AA, DHA, ω-3 fatty acids; a-tocopherol; γ-tocopherol, vitamin E, CoQ10; and β-carotene to said pregnant woman to reach a ratio corresponding to a ratio of a control population;

wherein said control population is a population of pregnant women characterized by normal blood pressure and absence of: obesity, diabetes, urine protein excretion and medical complications.

29. The method of claim 28, wherein said lipid is selected from: AA, DHA, an ω-3 fatty acid, a-tocopherol, CoQ10 and β-carotene; and said amount is effective to establish a ratio selected from the group consisting of:

(±)5-8, 12-iso-iPF_2a-VI (pg) / β-carotene (μΜ);

(±)5-8, 12-iso-iPF_2a-VI (pg) / (β-carotene / % ω-3);

(±)5-8, 12-iso-iPF_2a-VI (pg) / (β-carotene / Ul ω-3);

(±)5-8, 12-iso-iPF_2a-VI (pg) / (β-carotene / PI ω-3);

(±)5-8, 12-iso-iPF_2a-VI (pg) / (Ul ω-3 / (CoQ10 / β-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) / (% ω-3 / (CoQ10 / β-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) / (Ul ω-3/ (a-tocopherol /CoQ10 ^-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) / (% ω-3 / (a-tocopherol /CoQ10 ^-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) / (DHA / (a-tocopherol /CoQ10 ^-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) / (AA / (a-tocopherol /CoQ10 ^-carotene ));

(±)5-8, 12-iso-iPF_2a-VI (pg) x BMI / (Ul ω-3 / (a-tocopherol /CoQ10 ^-carotene )); corresponding to a control population;

wherein BMI is body mass index, Ul ω-3 is an unsaturation index for omega-3 fatty acids corresponding to the formula: (%Monoenoic x 1) + (%Dienoic x 2) + (%Trienoic x 3) + (%Tetraenoic x 4) + (%Pentaenoic x 5) + (%Hexaenoic x 6); said omega-3 fatty acids being: C18:3w3 and C18:4w3 and C20:3w3 and C20:4w3 and C20:5w3 and C22:5w3 and C22:6w3.

30. The method of claim 28, wherein said lipid is selected from: an ω-3 fatty acid, a- tocopherol, CoQ10 and β-carotene; and said amount is effective to establish a ratio of:

(±)5-8.12-iso-iPF2a-VI fpq) x BMI

(Ul ω-3 / (a-tocopherol /CoQ10 /p-carotene )) corresponding to a control population ratio.