Michaela Wirtz

Tissue-Specific Effects of In Vitro Fertilization Procedures on Genomic Cytosine Methylation Levels in Overgrown and Normal Sized Bovine Fetuses

Abstract Epigenetic perturbations are assumed to be responsible for phenotypic abnormalities of fetuses and offspring originating from in vitro embryo techniques. We studied 29 viable Day-80 bovine fetuses to assess the effects of two in vitro fertilization protocols (IVF1 and IVF2) on fetal phenotype and genomic cytosine methylation levels in liver, skeletal muscle, and brain. The IVF1 protocol employed 0.01 U/ml of FSH and LH in oocyte maturation medium and 5% estrous cow serum (ECS) in embryo culture medium, whereas the IVF2 protocol employed 0.2 U/ml of FSH and no LH for oocyte maturation and 10% ECS for embryo culture. Comparisons with in vivo–fertilized controls (n = 14) indicated an apparently normal phenotype for IVF1 fetuses (n = 5), but IVF2 fetuses (n = 10) were significantly heavier (19.9%) and longer (4.7%), with increased heart (25.2%) and liver (27.9%) weights, and thus displayed an overgrowth phenotype. A clinicochemical screen of 18 plasma parameters revealed significantly increased levels of insulin-like growth factor 1 (40.8%) and creatinine (37.5%) in IVF2, but not in IVF1, fetuses. Quantification of genomic 5-methylcytosine (5mC) by capillary electrophoresis indicated that both IVF1 and IVF2 fetuses differed from controls. We observed significant DNA hypomethylation in liver and muscle of IVF1 fetuses (−16.1% and −9.3%, respectively) and significant hypermethylation in liver of IVF2 fetuses (+11.2%). The 5mC level of cerebral DNA was not affected by IVF protocol. Our data indicate that bovine IVF procedures can affect fetal genomic 5mC levels in a protocol- and tissue-specific manner and show that hepatic hypermethylation is associated with fetal overgrowth and its correlated endocrine changes.

Analysis of special surfactants by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry

Multidimensional gas-chromatographic analyses of olesochemically based nonionic, anionic and several cationic surfactants in industrial cleaners are demonstrated. Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry allows the simultaneous determination of fatty alcohols, fatty alcohol sulphates and alkyl polyglucosides. In addition, the determination of fatty alcohol ethoxylates up to C(10)EO(8) (highest degree of ethoxylation) and C(18)EO(5) (longest C-chain at an ethoxylation degree of five) and the analysis of fatty alcohol alkoxylates that contain ethoxy (EO) and propoxy (PO) groups could be realized. Because of decomposition in the injector and a weak EI-fragmentation, cationic surfactants such as alkyl benzyl dimethyl ammonium chloride could also be identified by their characteristic fragments. Thermogravimetric analyses confirmed that the temperature in a normal GC injector is not high enough to cause thermal decomposition of esterquats. However, we could demonstrate that a modified silylation procedure forms decomposition products of esterquats in the GC injector which are detectable by GCxGC-(TOF)MS and allows the identification of such GC-atypical analytes.

Quantitative matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis of synthetic polymers and peptides.

Matrix-assisted laser desorption ionization (MALDI) is a very powerful and widely used mass spectrometric technique to ionize high molecular weight compounds. The most commonly used dried droplet (DD) technique can lead to a concentration distribution of the analyte on the target and is therefore often not suitable for reproducible analyses. We developed a new solvent-free deposition technique, called compressed sample (CS), to prevent the distribution of the analytes caused by the crystallization of the compounds. The CS technique presented in this work allows the quantitative analysis of synthetic polymers such as derivatized maltosides with correlation coefficients of 0.999 and peptides up to 3500 Da with correlation coefficients of at least 0.982 without the use of stable-isotope-labeled standards.

Combination of chemical and electron-impact ionisation with GC×GC–qMS for characterization of fatty alcohol alkoxylate polymers in the low-molecular-weight range up to 700 Da

The class of fatty alcohol alkoxylates describes surfactants that are synthesised by reaction of fatty alcohols with alkoxides such as ethylene oxide or propylene oxide or a combination of both as copolymers. Such alkoxylates are used, for example, as nonionic surfactants in home and industrial cleaning and washing agents. Chemical characteristics of such alkoxylate copolymers, for example the degree of alkoxylation, the arrangement of building blocks (random or block polymerisation), the type of the starter, and endcapping, play an important role in application behaviour. The analysis of these characteristics is challenging because in many cases such copolymers have high polydispersity and a large number of constitutional isomers depending on the degree of alkoxylation. Furthermore, the alkoxylates often occur in a complex multicomponent matrix. Here we present a method for characterization of silylated fatty alcohol alkoxylates in the low-molecular-weight range by means of comprehensive two-dimensional gas chromatography–mass spectrometry with electron impact and chemical ionisation. This method also enables detailed analysis of the alkoxylates in a complex matrix such as modern detergents.