Consuelo Cháfer-Pericás

Ultra high performance liquid chromatography coupled to tandem mass spectrometry determination of lipid peroxidation biomarkers in newborn serum samples

Byproducts of arachidonic (AA) and docosahexaenoic acid (DHA) oxidation are highly relevant for the study of free radical associated conditions in the perinatal period. Plasma metabolites can provide the clinician with a snapshot of the oxidant status of patients before and after specific clinical interventions (e.g.: supplementation with oxygen). We describe a new andreliable ultra-performance liquid mass spectrometry method to determine F2-isoprostanes and other byproducts (isoprostanes, isofurans, neuroprostanes, neurofurans) in newborn serum samples. Cord blood samples were obtained from severely depressed newborn infants (Apgar score 1 min < 3; arterial cord pH < 7.00), and aliquoted for serum determination and stored at -80 °C. A UHPLC-MS/MS method was employed. It has a series of technical advantages: simple sample treatment; reduced sample volume (100 μL) which is essential for preterm neonates with low circulating blood volume, high throughput of sample analysis (96 samples in less than 24 h) and high selectivity for different isoprostanes isomers. Excellent sensitivity was achieved within limits of detection between 0.06 and 4.2 nmol L(-1), which renders this method suitable to monitoranalyte concentration in newborn samples. The methods precision was satisfactory; with coefficients of variation around 5-12% (intra-day) and 7-17% (inter-day). The reliability of the described method was assessed by analysis of spiked serum samples obtaining recoveries between 70% and 120%. The proposed method has rendered suitable for serum determination for newborn babies at risk of oxygen free radical associated conditions.

Potential oxidative stress biomarkers of mild cognitive impairment due to Alzheimer disease

The high and increasing incidence of Alzheimer Disease (AD) worldwide is a major global concern. Classical diagnosis is carried out in the dementia phase, often in the moderate stages when treatment efficacy is limited. Nowadays, early diagnosis, even in pre-dementia stages, is possible in selected cases within an appropriate clinical setting, employing cerebral spinal fluid (CSF) sample analysis and neuroimaging procedures. In spite of the accurate diagnosis achieved by novel CSF biomarkers or positron emission tomography beta-amyloid tracers, these tests are invasive and expensive. Therefore, important work is being carried out to discover reliable biomarkers in peripheral biofluids (blood, plasma, urine) to be incorporated in clinical routine for early AD diagnosis. Although the nature of AD pathogenesis is complex, it is known that oxidative stress plays a key role, for which biomarkers are easily determined in peripheral biofluids. This review summarizes recent research on oxidative stress biomarkers in mild cognitive impairment due to AD. Among them, a promising research line is the study of the relationship between lipid peroxidation biomarkers and early AD clinical features. Results show a pronounced imbalance between scientific production and clinical reality due to the lack of clinical validation. We conclude that an important field in oxidative stress biomarkers could be developed with the aim to help clinicians in early disease diagnosis, effective treatment initiation and reliable disease monitoring.

Development of a reliable analytical method to determine lipid peroxidation biomarkers in newborn plasma samples

This paper describes a reliable analytical method based on ultra-performance liquid chromatography coupled to tandem mass spectrometry to determine F2-isoprostanes and other total byproducts (isoprostanes, isofurans, neuroprostanes and neurofurans) as lipid peroxidation biomarkers in newborn plasma samples. The proposed procedure is characterized by a simple sample treatment employing a reduced sample volume (100µL). Also, it shows a high throughput and high selectivity to determine simultaneously different isoprostane isomers in a large number of samples. The reliability of the described method was demonstrated by analysis of spiked plasma samples, obtaining recoveries between 70% and 130% for most of the analytes. Taking into account the implementation of further clinical studies, it was demonstrated the proper sensitivity of the method by means of the analysis of few human newborn plasma samples. In addition to this, newborn piglet plasma samples (n=80) were analyzed observing that the developed method was suitable to determine the analyte levels present in this kind of samples. Therefore, this analytical method could be applied in further clinical research about establishment of reliable lipid peroxidation biomarkers employing this experimental model.

Development of a reliable method based on ultra-performance liquid chromatography coupled to tandem mass spectrometry to measure thiol-associated oxidative stress in whole blood samples

The aminothiols are biological compounds with numerous vital functions. One of the most relevant is their role as antioxidant systems. The reduced to oxidized ratios are extremely useful indicators of oxidative stress and cellular redox status. We have validated an ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method to determine the levels of glutathione, cysteine, homocysteine, and their respective oxidized compounds in whole blood samples. Results showed excellent linearity for all the analytes with correlation coefficients between 0.990 and 0.997, suitable precision with intra-day coefficient of variation ≤20%, and satisfactory accuracy with recoveries between 75 and 130%. The limits of detection in whole blood samples were 1.16 nmol L(-1) for glutathione, 115.8 nmol L(-1) for oxidized glutathione, 9.3 nmol L(-1) for homocystine, 92.6 nmol L(-1) for homocysteine, 347 nmol L(-1) for cystine and 0.23 nmol L(-1) for cysteine. The suitability of the method was ascertained in whole blood samples (n=80) from a consolidated experimental model of hypoxia-reoxygenation in newborn piglets.

Protein-bound tyrosine oxidation, nitration and chlorination by-products assessed by ultraperformance liquid chromatography coupled to tandem mass spectrometry

BACKGROUND Free radicals cause alterations in cellular protein structure and function. Oxidized, nitrated, and chlorinated modifications of aromatic amino acids including phenylalanine and tyrosine are reliable biomarkers of oxidative stress and inflammation in clinical conditions. OBJECTIVE To develop, validate and apply a rapid method for the quantification of known hallmarks of tyrosine oxidation, nitration and chlorination in plasma and tissue proteins providing a snapshot of the oxidative stress and inflammatory status of the organism and of target organs respectively. MATERIAL AND METHODS The extraction and clean up procedure entailed protein precipitation, followed by protein re-suspension and enzymatic digestion with pronase. An Ultra Performance Liquid Chromatography-tandem Mass Spectrometry (UPLC-MS/MS) method was developed to quantify protein released ortho-tyrosine (o-Tyr), meta-tyrosine (m-Tyr), 3-nitrotyrosine (3NO2-Tyr) and 3-chlorotyrosine (3Cl-Tyr) as well as native phenylalanine (Phe) and tyrosine (p-Tyr) in plasma and tissue from a validated hypoxic newborn piglet experimental model. RESULTS In plasma there was a significant increase in the 3NO2-Tyr/p-Tyr ratio. On the other hand m-Tyr/Phe and 3Cl-Tyr/p-Tyr ratios were significantly increased in liver of hypoxic compared with normoxic animals. Although no significant differences were found in brain tissue, a clear tendency to increased ratios was observed under hypoxic conditions. CONCLUSIONS UPLC-MS/MS has proven suitable for the analysis of plasma and tissue samples from newborn piglets. The analysis of biomarkers of protein oxidation, nitration and chlorination will be applied in future studies aiming to provide a deeper insight into the mechanisms of oxidation-derived protein modification caused during neonatal asphyxia and resuscitation.

Preliminary case control study to establish the correlation between novel peroxidation biomarkers in cord serum and the severity of hypoxic ischemic encephalopathy

BACKGROUND Hypoxic-ischemic encephalopathy (HIE) has deleterious neurological consequences. To identify patients at risk of neuronal damage deserving implementation of neuroprotective strategies clinicians have relied on prenatal sentinel events, postnatal clinical assessment (Apgar score), and blood gas analysis. This feasibility study aimed to assess if lipid peroxidation byproducts associated with neuronal damage correlated with cord blood metabolic acidemia in patients with HIE. POPULATION AND METHODS This is a case/control study in which cases were newborn infants with severe acidemia (pH<7.00; base excess ≥12mmol/L) while control babies exhibited normal gases (pH=7.20-7.40; base excess=-4 to +4mmol/L) in the first cord blood analysis performed immediately after birth. Concomitantly, lipid peroxidation byproducts were determined using ultra performance liquid chromatography coupled to mass spectrometry in the same cord blood sample. RESULTS A total of 19 controls and 20 cases were recruited. No differences in gestational characteristics were present. However, cases exhibited profound metabolic alterations as compared to controls (Cases vs. CONTROL pH=6.90±0.1 vs. 7.33±0.03; base excess=-15±3 vs. -1±2mmol/L), 85% were admitted to the NICU, and 50% developed symptoms of HIE. 8-iso-15(R)-PGF2α (P=0.01) and total isoprostanes (P=0.045) presented statistically significant differences between cases and control groups and correlated with level of HIE. CONCLUSIONS The 8-iso-15(R)-PGF2α and isoprostanes reflecting oxidative damage are significantly increased in severe postnatal acidemia. Follow up studies with adequate power are necessary to confirm if these biomarkers measured in cord blood serum could be predictive of neonatal encephalopathy.

Oxygen Supplementation to Stabilize Preterm Infants in the Fetal to Neonatal Transition: No Satisfactory Answer

Fetal life elapses in a relatively low oxygen environment. Immediately after birth with the initiation of breathing, the lung expands and oxygen availability to tissue rises by twofold, generating a physiologic oxidative stress. However, both lung anatomy and function and the antioxidant defense system do not mature until late in gestation, and therefore, very preterm infants often need respiratory support and oxygen supplementation in the delivery room to achieve postnatal stabilization. Notably, interventions in the first minutes of life can have long-lasting consequences. Recent trials have aimed to assess what initial inspiratory fraction of oxygen and what oxygen targets during this transitional period are best for extremely preterm infants based on the available nomogram. However, oxygen saturation nomogram informs only of term and late preterm infants but not on extremely preterm infants. Therefore, the solution to this conundrum may still have to wait before a satisfactory answer is available.

Reference ranges for cortisol and α-amylase in mother and newborn saliva samples at different perinatal and postnatal periods.

This paper describes a reliable analytical method based on Ultra High-Performance Liquid Chromatography coupled to tandem mass spectrometry to determine cortisol in saliva samples from healthy mothers (n=87) and newborns (n=65) at different time points: (i) 38 weeks of gestation, (ii) in the immediate postnatal period (48h) after a term delivery and, (iii) 3 months after delivery. The procedure is characterized by a simple sample treatment employing a sample volume of 25μL. In addition to this, salivary α-amylase was determined using a commercial kit. We have proposed potential reference ranges in saliva for cortisol (0.7-35nmolL(-1)) and α-amylase (2-500UmL(-1)) in mothers, and for cortisol (0.1-56nmolL(-1)) and α-amylase (0.1-500UmL(-1)) in newborn infants. In addition, statistical differences between the two sensitive population groups (mothers and newborns) at the perinatal and postnatal periods were studied. A lower concentration for maternal cortisol was found at 38 weeks of gestation than at 48h (p=0.048) or 3 months after delivery (p=0.021). Similar results were found for the α-amylase determinations. Hence, higher concentrations than could be expected from a chronic stress marker were found at 3 months after delivery than at 38 weeks of gestation (p<0.001) or 48h after delivery (p<0.001). We conclude that this analytical method could be applied to further clinical research on perinatal and postnatal stress, such as threatened preterm labor and/or parenting stress, respectively.

Can stress biomarkers predict preterm birth in women with threatened preterm labor

BACKGROUND Preterm birth is a major paediatric challenge difficult to prevent and with major adverse outcomes. Prenatal stress plays an important role on preterm birth; however, there are few stress-related models to predict preterm birth in women with Threatened Preterm Labor (TPL). OBJECTIVE The aim of this work is to study the influence of stress biomarkers on time until birth in TPL women. METHODS Eligible participants were pregnant women between 24 and 31 gestational weeks admitted to the hospital with TPL diagnosis (n=166). Stress-related biomarkers (α-amylase and cortisol) were determined in saliva samples after TPL diagnosis. Participants were followed-up until labor. A parametric survival model was constructed based on α-amylase, cortisol), TPL gestational week, age, parity, and multiple pregnancy. The model was adjusted using a logistic distribution and it was implemented as a nomogram to predict the labor probability at 7- and 14-day term. RESULTS The time until labor was associated with cortisol (p=0.001), gestational week at TPL diagnosis (p=0.004), and age (p=0.02). Importantly, high cortisol levels at TPL diagnosis were predictive of latency to labor. Validation of the model yielded an optimum corrected AUC value of 0.63. CONCLUSIONS High cortisol levels at TPL diagnosis may have an important role in the preterm birth prediction. Our statistical model implemented as a nomogram provided accurate predictions of individual prognosis of pregnant women.

A preliminary study to assess the impact of maternal age on stress-related variables in healthy nulliparous women

PURPOSE Childbearing age has progressively increased in industrialized countries. The impact of this delay on motherhood, however, requires further research. METHODS The study sample included a prospective cohort of healthy nulliparous pregnant women aged between 18 and 40 years (n=148) assessed at 38 weeks gestation (Time#1, T1), 48h after birth (Time#2, T2), and 3 months after birth (Time#3, T3). The effect of age on psychological, biological, and social variables was evaluated. Maternal psychological symptoms in terms of depression and anxiety were assessed at T1-T3; and parenting stress at T3. Stress biomarkers (cortisol, α-amylase) were determined in mothers at T1-T3. Questionnaires addressing social functioning (i.e., family functioning, maternal attitudes, and social support) were conducted at T3. Bayesian additive models were used to analyze the data. RESULTS Depressive symptoms showed a steep increase starting from 35 years of age at T1 and an U-shaped relationship with a minimum around 30 years old at T3. The same results were observed for parenting stress. Cortisol levels increased sharply from 30 years of age at T3. Family functioning, maternal attitudes, and social support improved moderately from 30 years of age. CONCLUSIONS Prenatal depressive symptoms were higher in older women, but postpartum depressive symptoms and parenting stress increased in both younger and older women. Nevertheless, cortisol levels just increased in older ages at postpartum. In contrast, social functioning (family functioning, maternal attitudes, and social support) improved with age. We conclude that these social advantages may compensate for other disadvantages of delayed childbearing (i.e., depressive symptoms, parenting stress, and high cortisol level).