Prediction of preterm delivery by second trimester inflammatory biomarkers in the amniotic fluid

ObjectiveTo search for a correlation between mid-pregnancy altered levels of inflammatory markers and preterm delivery.MethodsA prospective cohort series included 39 patients undergoing amniocentesis one additional milliliter of amniotic fluid (AF) was stored for later dosage of interleukin-6 (Il-6), matrix metalloproteinase-9 (MMP-9), glucose and C-reactive protein (CRP). Maternal serum CRP and glucose levels were also obtained. Exclusion criteria were multiple pregnancies, chorioamnionitis, group B streptococcus colonization, bacterial vaginosis and cases with proven aneuploidy. We searched for correlation between AF and plasmatic markers and also for a difference between patients with term and preterm delivery.Results33 participants were eligible and one third had preterm delivery. Levels of the plasmatic biomarkers did not correlate with the AF biomarkers except for plasmatic glucose and AF IL-6 levels (r = 0.350; p = 0.016). The levels of all AF biomarkers did not differ significantly between the pre-term and the term groups (p > 0.05). The optimal screening cutoffs for identifying pregnancies at risk were different than the ones initially indicated.ConclusionMid-pregnancy amniotic fluid biomarker levels do not correlate with preterm delivery. Plasma CRP is not correlated with these markers. Cutoff levels suggested are sparse and heterogeneous. Larger studies are needed before advising routine measurement of these markers.     

Metabolic biomarkers of prenatal disorders: an exploratory NMR metabonomics study of second trimester maternal urine and blood plasma

This work describes an exploratory NMR metabonomic study of second trimester maternal urine and plasma, in an attempt to characterize the metabolic changes underlying prenatal disorders and identify possible early biomarkers. Fetal malformations have the strongest metabolic impact in both biofluids, suggesting effects due to hypoxia (leading to hypoxanthine increased excretion) and a need for enhanced gluconeogenesis, with higher ketone bodies (acetone and 3-hydroxybutyric acid) production and TCA cycle demand (suggested by glucogenic amino acids and cis-aconitate overproduction). Choline and nucleotide metabolisms also seem affected and a distinct plasma lipids profile is observed for mothers with fetuses affected by central nervous system malformations. Urine from women who subsequently develop gestational diabetes mellitus exhibits higher 3-hydroxyisovalerate and 2-hydroxyisobutyrate levels, probably due to altered biotin status and amino acid and/or gut metabolisms (the latter possibly related to higher BMI values). Other urinary changes suggest choline and nucleotide metabolic alterations, whereas lower plasma betaine and TMAO levels are found. Chromosomal disorders and pre-preterm delivery groups show urinary changes in choline and, in the latter case, in 2-hydroxyisobutyrate. These results show that NMR metabonomics of maternal biofluids enables the noninvasive detection of metabolic changes associated to prenatal disorders, thus unveiling potential disorder biomarkers.     

Metabolic profiling of human urine by CE-MS using a positively charged capillary coating and comparison with UPLC-MS

The potential of capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS) using capillaries coated with a triple layer of polybrene-dextran sulfate-polybrene (PB-DS-PB) was evaluated for metabolic profiling of human urine. The method covers various metabolite classes and stable metabolic profiles of urine samples were obtained with favourable migration time repeatability (RSDs <1%). The PB-DS-PB CE-TOF-MS method was used for the analysis of human urine samples from 30 males and 30 females, which had been previously analyzed by reversed-phase UPLC-TOF-MS. Multivariate data analysis of the obtained data provided clear distinction between urine samples from males and females, emphasizing gender differences in metabolic signatures. Nearly all compounds responsible for male-female classification in CE-TOF-MS were different from the classifying compounds in UPLC-TOF-MS. Almost all compounds causing classification in the CE-TOF-MS study were highly polar and did not exhibit retention in the reversed-phase UPLC system. In addition, the CE-TOF-MS classifiers had an m/z value in the range of 50-150, whereas 95% of the classifying features found with UPLC-TOF-MS had an m/z value above 150. The CE-TOF-MS method therefore appears to be highly complementary to the UPLC-TOF-MS method providing classification based on different classes of metabolites.     

Direct and rapid mass spectral fingerprinting of maternal urine for the detection of Down syndrome pregnancy

Background

The established methods of antenatal screening for Down syndrome are based on immunoassay for a panel of maternal serum biomarkers together with ultrasound measures. Recently, genetic analysis of maternal plasma cell free (cf) DNA has begun to be used but has a number of limitations including excessive turn-around time and cost. We aimed to develop an alternative method based on urinalysis that is simple, affordable and accurate.

Method

101 maternal urine samples sampled at 12–17 weeks gestation were taken from an archival collection of 2567 spot urines collected from women attending a prenatal screening clinic. 18 pregnancies in this set subsequently proved to be Down pregnancies. Samples were either neat urine or diluted between 10 to 1000 fold in dH₂O and subjected to matrix assisted laser desorption ionization (MALDI), time of flight (ToF) mass spectrometry (MS). Data profiles were examined in the region 6,000 to 14,000 m/z. Spectral data was normalised and quantitative characteristics of the profile were compared between Down and controls.

Results

In Down cases there were additional spectral profile peaks at 11,000-12,000 m/z and a corresponding reduction in intensity at 6,000-8,000 m/z. The ratio of the normalised values at these two ranges completely separated the 8 Down syndrome from the 39 controls at 12–14 weeks. Discrimination was poorer at 15–17 weeks where 3 of the 10 Down syndrome cases had values within the normal range.

Conclusions

Direct MALDI ToF mass spectral profiling of maternal urinary has the potential for an affordable, simple, accurate and rapid alternative to current Down syndrome screening protocols.     

Advances in the proteomics of amniotic fluid to detect biomarkers for chromosomal abnormalities and fetomaternal complications during pregnancy

Introduction: Amniotic fluid (AF) is a dynamic and complex mixture that reflects the physiological condition of developing fetus. In the last decade, proteomic analysis of AF for 16–18 weeks normal pregnancy has been done for the composition and functions of this fluid. Other body fluids such as urine, sweat, tears, etc. are being used for diagnosis of disease, but an insight into protein biomarkers of amniotic fluid can save the fetus and mother from future complications.

Areas covered: We have covered the proteomics of amniotic fluid done since 2000, in order to strengthen the establishment of these techniques as a recognized diagnostic tool in the field. After classifying the diseases based on chromosomal aneuploidies, gestational changes, and inflammation caused during pregnancy; we have focused on amniotic fluid to detect various complications during and post pregnancy and its effect on the fetomaternal relationship.

Expert comment: The main protein biomarkers responsible for various syndromes, diseases, and complications have been summarized. Major proteins identified for gestational conditions are IGFBP-1, fibrinogen, neutrophil defensins like calgranulins A and C, cathelicidin, APOA1, TRFE, etc. Validation of particular technique and establishing a single standardized biomarker for the diagnosis to avoid any overlapping for different diseases is required. After certain improvements, proteomics approach can be considered for diagnosis of diseases associated with fetal-maternal health.     

NMR spectroscopy based metabolic profiling of urine and serum for investigation of physiological perturbations during radiation sickness

Radiation accidents are rare events that induce radiation syndrome, a complex pathology which is difficult to treat. In medical management of radiation victims, life threatening damage to different physiological systems should be taken into consideration. The present study was proposed to identify metabolic and physiological perturbations in biofluids of mice during different phases of radiation sickness using ¹H nuclear magnetic resonance (¹H NMR) spectroscopy and pattern recognition (PR) technique. The ¹H NMR spectra of the biofluids collected from mice irradiated with 5 Gray (Gy) at different time points during radiation sickness were analysed visually and by principal components analysis. Urine and serum spectral profile clearly showed altered metabolic profiles during different phases of radiation sickness. Increased concentration of urine metabolites viz. citrate, α ketoglutarate, succinate, hippurate, and trimethylamine during prodromal and clinical manifestation phase of radiation sickness shows altered gut microflora and energy metabolism. On the other hand, serum nuclear magnetic resonance (NMR) spectra reflected changes associated with lipid, energy and membrane metabolism during radiation sickness. The metabonomic time trajectory based on PR analysis of ¹H NMR spectra of urine illustrates clear separation of irradiated mice group at different time points from pre dose. The difference in NMR spectral profiles depicts the pathophysiological changes and metabolic disturbances observed during different phases of radiation sickness, that in turn, demonstrate involvement of multiple organ dysfunction. This could further be useful in development of multiparametric approach for better evaluation of radiation damage as well as for medical management during radiation sickness.     

A gender-specific discriminator in Sprague-Dawley rat urine: the deployment of a metabolic profiling strategy for biomarker discovery and identification

The use of nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) as complementary analytical techniques for open metabolic profiling is illustrated in the context of defining urinary biochemical discriminators between male and female Sprague-Dawley rats. Subsequent to the discovery of a female-specific urinary discriminator by LC-MS, further LC, MS, and NMR methods have been applied in a coordinated effort to identify this urinary component. Thereafter, the biological relevance and context of the identified component, in this case a steroid metabolite, has been achieved. This approach will be deployed in future studies of disease, drug efficacy, and toxicity to discover and identify biologically relevant markers.     

Metabonomic and metallomic profiling in the amniotic fluid of malnourished pregnant rats

Epidemiology and studies in animal models have revealed that prenatal malnutrition is highly correlated with abnormal fetal neurodevelopment. We present here a combined metabonomic and metallomic profiling technique to associate the metabolic and trace-elemental composition variations of rat amniotic fluid (AF) in malnourished pregnant rats with the retardation of fetal rat neurodevelopment. The AF samples from three groups of pregnant Sprague-Dawley rats, which were fed either a normal diet, a low-protein diet, or "a famine diet", were subjected to GC/MS and ICP/MS combined with multivariate data analysis (MVDA). PCA scores plot of both GC/MS and ICP/MS data showed similar and unique metabolic signatures of AF in response to the different diets. Rats in the famine group released increased amounts of glycine, inositol, putrescine, and rubidium and decreased amounts of methionine, dopa, tryptophan, glutamine, zinc, cobalt, and selenium in the AF. These discriminable variations in the AF may indicate the abnormality of a number of metabolic pathways in fetal rats including the folate cycle and methionine pathway, the monoamine pathway, and tri-iodothyronine (T3) metabolism. The abnormalities may be the result of metabolites or elemental differences or a combination of both. This study demonstrates the potential of combining profiling of small-molecule metabolites and trace elements to broaden the understanding of biological variations associated with fetal neurodevelopment induced by environmental perturbation.     

Zinc and copper of fetal organs during the second trimester of pregnancy

In fetus with a mean gestational age of 18 weeks (range 15-25, n = 14), zinc and copper concentrations in liver, femur, rib, and skeletal muscle were measured. Zinc and copper concentrations are highest in liver. A trend of decreasing liver zinc concentrations during gestational age is suggested. Zinc concentrations are significantly correlated with copper concentrations in liver and in femur, suggesting steady growth in both organs. Femur zinc values rank ca. 30% of those in liver, femur copper, ca. 2%. Zinc or copper concentrations in rib are of the same levels as in skeletal muscle. Their concentration for zinc ranks ca. 20%, for copper, ca. 5% of the values in liver. All zinc and copper values are lower than reported in third trimester fetal organs. Calculated zinc/copper molar ratios are distinctive for the various organs: in liver, 6 +/- 1, in femur, 73 +/- 8, and in soft tissues, 26 +/- 3. Calculated ratios from published values obtained from the third trimester of pregnancy show that the ratios in liver and skeletal muscle maintain these levels. The zinc/copper molar ratio can serve as an internal reference in zinc and/or copper measurements.     

Comparison of 1D and 2D NMR spectroscopy for metabolic profiling

High-resolution, liquid state nuclear magnetic resonance (NMR) spectroscopy is a popular platform for metabolic profiling because the technique is nondestructive, quantitative, reproducible, and the spectra contain a wealth of biochemical information. Because of the large dynamic range of metabolite concentrations in biofluids, statistical analyses of one-dimensional (1D) proton NMR data tend to be biased toward selecting changes in more abundant metabolites. Although two-dimensional (2D) proton-proton experiments can alleviate spectral crowding, they have been mainly used for structural determination. In this study, 2D total correlation spectroscopy NMR was used to compare the global metabolic profiles of urine obtained from wild-type and Abcc6-knockout mice. The 2D data were compared to an improved 1D experiment in which signal contributions from macromolecules and the urea peak have been spectroscopically removed for more accurate quantitation of low-abundance metabolites. Although statistical models from both 1D and 2D data could differentiate samples acquired from the two groups of mice, only the 2D spectra allowed the characterization of statistically relevant changes in the low-abundance metabolites. While acquisition of the 2D data require more time, the data obtained resulted in a more meaningful and comprehensive metabolic profile, aided in metabolite identifications, and minimized ambiguities in peak assignments.     

Transport and storage of amniotic fluid samples for prenatal diagnosis of metabolic diseases

Summary 1. A method for freezing uncultured amniotic fluid cells is presented, which allows their use as pregnancy-age matched controls in prenatal diagnosis of metabolic diseases. 2. Amniotic fluid cells were successfully cultured after up to 7 days in transport, which makes prenatal diagnosis available to parents living a long way from specialized centers.

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Zusammenfassung 1. Eine Einfriermethode für nichtkultivierte Fruchtwasserzellen wird beschrieben, wodurch man diese wie Kontrollmuster anwenden kann bei pr?nataler Diagnostik von Stoffwechselkrankheiten. 2. Fruchtwasserzellen wurden erfolgreich kultiviert nach langzeitigem Transport (7 Tage); dieser Befund bringt eine pr?natale Diagnostik im Bereich entfernt von spezialisierten Zentren lebender Eltern.

     

A comprehensive peptidome profiling technology for the identification of early detection biomarkers for lung adenocarcinoma

The mass spectrometry-based peptidomics approaches have proven its usefulness in several areas such as the discovery of physiologically active peptides or biomarker candidates derived from various biological fluids including blood and cerebrospinal fluid. However, to identify biomarkers that are reproducible and clinically applicable, development of a novel technology, which enables rapid, sensitive, and quantitative analysis using hundreds of clinical specimens, has been eagerly awaited. Here we report an integrative peptidomic approach for identification of lung cancer-specific serum peptide biomarkers. It is based on the one-step effective enrichment of peptidome fractions (molecular weight of 1,000-5,000) with size exclusion chromatography in combination with the precise label-free quantification analysis of nano-LC/MS/MS data set using Expressionist proteome server platform. We applied this method to 92 serum samples well-managed with our SOP (standard operating procedure) (30 healthy controls and 62 lung adenocarcinoma patients), and quantitatively assessed the detected 3,537 peptide signals. Among them, 118 peptides showed significantly altered serum levels between the control and lung cancer groups (p<0.01 and fold change >5.0). Subsequently we identified peptide sequences by MS/MS analysis and further assessed the reproducibility of Expressionist-based quantification results and their diagnostic powers by MRM-based relative-quantification analysis for 96 independently prepared serum samples and found that APOA4 273-283, FIBA 5-16, and LBN 306-313 should be clinically useful biomarkers for both early detection and tumor staging of lung cancer. Our peptidome profiling technology can provide simple, high-throughput, and reliable quantification of a large number of clinical samples, which is applicable for diverse peptidome-targeting biomarker discoveries using any types of biological specimens.     

Biological action and half life in plasma of intramuscular sulprostone for termination of second trimester pregnancy

Sulprostone levels in plasma, platelet function and clinical effects were examined in four patients after intramuscular injection of 500 μg sulprostone for induction of missed and therapeutic abortion in the second trimester of pregnancy. The half life of sulprostone in plasma was 34 (30–45) minutes. The highest sulprostone levels were found 10 to 20 minutes after injection (0.25–0.77 ng/ml) and after 120–240 minutes the values in plasma were no longer distinguishable from zero. Platelet count remained constant. 20 minutes after the injection platelet aggregation was slightly decreased, but not to a significant level. There was no correlation between sulprostone levels in plasma and pain of labour or induction abortion times. Side effects were minimal.     

1H NMR metabolic profiling of human serum associated with benign and malignant gallstone diseases

Proton NMR based metabolic profile of serum associated with different gallbladder pathologies is presented. Quantitative and qualitative variations in the metabolic profile of serum in control samples and three different pathologies of gallbladder, chronic cholecystitis, xanthogranulomatous cholecystitis and carcinoma of gallbladder has been evaluated by use of ¹H NMR based metabonomics and multivariate chemometric methods. Multivariate partial least square discriminant analysis of ¹H NMR spectra showed a clear discrimination between control and diseased groups on the basis of quantitative and qualitative metabolic variations. Increased levels of lactate and pyruvate whereas decreased levels of glucose, some amino acids and low density lipoprotein/very low density lipoprotein (LDL/VLDL) were observed. These metabolites, responsible for class discrimination, from different metabolic pathways could be considered as the signatures of the carcinoma of gallbladder.