Manja Wölter

A proteome signature for intrauterine growth restriction derived from multifactorial analysis of mass spectrometry‐based cord blood serum profiling

Intrauterine growth restriction (IUGR) is defined as a condition in which the fetus does not reach its genetically given growth potential, resulting in low birth weight. IUGR is an important cause of perinatal morbidity and mortality, thus contributing substantially to medically indicated preterm birth in order to prevent fetal death. We subjected umbilical cord blood serum samples either belonging to the IUGR group (n = 15) or to the control group (n = 15) to fractionation by affinity chromatography using a bead system with hydrophobic interaction capabilities. So prepared protein mixtures were analyzed by MALDI‐TOF mass spectrometric profiling. The six best differentiating ion signals at m/z 8205, m/z 8766, m/z 13 945, m/z 15 129, m/z 15 308, and m/z 16 001 were collectively assigned as IUGR proteome signature. Separation confidence of our IUGR proteome signature reached a sensitivity of 0.87 and a specificity of 0.93. Assignment of ion signals in the mass spectra to specific proteins was substantiated by SDS‐PAGE in conjunction with peptide mass fingerprint analysis of cord blood serum proteins. One constituent of this proteome signature, apolipoprotein C‐III0, a derivative lacking glycosylation, has been found more abundant in the IUGR cord blood serum samples, irrespective of gestational age. Hence, we suggest apolipoprotein C‐III0 as potential key‐marker of the here proposed IUGR proteome signature, as it is a very low‐density lipoprotein (VLDL) and high‐density lipoprotein (HDL) member and as such involved in triglyceride metabolism that itself is discussed as being of importance in IUGR pathogenesis. Our results indicate that subtle alterations in protein glycosylation need to be considered for improving our understanding of the pathomechanisms in IUGR.

Mass spectrometric profiling of cord blood serum proteomes to distinguish infants with intrauterine growth restriction from those who are small for gestational age and from control individuals.

Intrauterine growth restriction (IUGR) is a multifactorial condition in that the fetus does not reach its genetically given growth potential. Besides its contribution to perinatal mortality, it is a risk factor for cardiovascular and metabolic diseases later in life. The diagnosis is based on antenatal sonography, which allows differentiating between IUGR and fetuses that are small by constitution (small for gestational age [SGA]). Yet, neither a clinical nor a biochemical tool is available to confirm reliably the diagnosis of IUGR postnatally. Recently, we identified umbilical cord blood proteins of the apolipoprotein family by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with differential signal intensities between the IUGR group and a control group. We hypothesized that identified molecules have the potential to generate a proteome profile specific for IUGR. A total of 114 serum samples (42 from the IUGR group, 12 from the SGA group, and 60 from the control group) of the umbilical vein (99 samples) and umbilical artery (15 samples) were analyzed. Sample quality was estimated by determining the abundance of hemoglobin (hemolysis) and CXC-motif chemokines CXCL4 and CXCL7 (platelet activation). Samples passing the quality criteria were forwarded to multiplex apolipoprotein profiling. Assay performance was tested with the sample sets, resulting in a sensitivity of 0.91 and a specificity of 0.85 in the test set with venous blood samples. Arterial cord blood samples followed the trend (sensitivity, 0.67; specificity, 0.85). SGA samples grouped together with the control samples. We conclude that the proteome profiling signature is confirmatory to clinical surveillance with the potential to identify neonates with IUGR postnatally in low-birth weight babies born at uncertain gestational age when antenatal sonography data have not been recorded.

Maternal Apolipoprotein B100 Serum Levels are Diminished in Pregnancies with Intrauterine Growth Restriction and Differentiate from Controls

Intrauterine growth restriction, a major cause of fetal morbidity and mortality, is defined as a condition in which the fetus does not reach its genetically given growth potential. Screening for intrauterine growth restriction biomarkers in the mothers blood would be of great help for optimal pregnancy management and timing of delivery as well as for identifying fetuses requiring further surveillance during their infancies.

PP032. Apolipoprotein profiling in umbilical cord blood of intrauterine growth restricted (IUGR) neonates

INTRODUCTION Fetal umbilical cord HDL concentration is lower in IUGR neonates as compared to gestational age matched controls (CTRL). The causes by now are unknown. A full apolipoprotein analysis of cord blood might help in understanding the changes in lipid metabolism seen in IUGR. OBJECTIVE To characterize cord blood apolipoprotein profile of IUGR neonates. METHODS Serum of venous umbilical cord blood (15 IUGR vs. 15 CTRL) was analyzed by Multiple Reaction Monitoring (MRM). 15 different known apolipoproteins were profiled. HDL and LDL were measured by colorimetric methods in fetal cord blood and their corresponding mothers. RESULTS Fetal HDL (p<0.0001), ApoC1 (p<0.0001), and ApoE (p=0.0001) levels were lower in IUGR as compared to CTRL. Fetal HDL levels were positive correlated to ApoE, ApoC1, and ApoA2 (r=0.79, r=0.74, r=0.56). Fetal LDL levels were positive correlated to ApoB, ApoE, ApoA2, and ApoC3 (r=0.74, r=0.67, r=0.57, r=0.55). Maternal LDL concentrations correlated positive to fetal ApoC1, ApoC2, and LCAT-concentration (r=0.54, r=0.52, r=0.52). DISCUSSION The results underlines the relevance of ApoE in fetal development. Moreover, we speculate that maternal lipid profile has an impact on fetal lipid metabolisms as evidenced by the association of maternal LDL levels and fetal ApoC1, ApoC2, and LCAT concentrations. This observation requires further confirmation and is worth to be analyzed since it provides a mechanistic link for therapeutic options.

OS079. Fetal deglycosylated apolipoprotein C-III (Apo C-III0) concentration is altered in intrauterine growth restriction

INTRODUCTION We recently demonstrated that serum lipid levels are altered in growth restricted fetuses (IUGR) [1]. OBJECTIVES We now aimed to analyse the proteome profile of umbilical cord blood in order to gain a greater understanding about metabolic changes in IUGR fetuses. METHODS umbilical cord blood serum samples of IUGR (n=15) and of gestational age matched controls (CN; n=15) were subjected to fractionation by affinity chromatography using a bead system with hydrophobic interaction capabilities. So prepared protein mixtures were forwarded to MALDI-TOF mass spectrometric profiling. Assignment of ion signals in the mass spectra to specific proteins was substantiated by SDS-PAGE in conjunction with peptide mass fingerprint analysis. Concentrations of proteins of interest were additionally measured by ELISA. Statistical estimations were performed by Students t-test and calculation of Spearmans correlation coefficient. RESULTS MALDI mass spectra showed on average more than 60 protein ion signals between m/z 4000 and 25,000. The six best differentiating ion signals were found at m/z 8205, m/z 8766, m/z 13,945, m/z 15,129, m/z 15,308, and m/z 16,001. One of the constituent of this proteome signature is the deglycosylated form of apolipoprotein C-III, apo C-III0 (8766 m/z) that is known to prevent triglycerides from catabolism. While total Apo CIII concentration tended to be decreased (IUGR 22.54μg/mL SD 10.25. CN 29.9μg/mL SD 15.46. p=0.1355) calculated Apo C-III0 concentration levels has been found to be more abundant in the IUGR cord blood serum samples (IUGR 1.99μg/mL SD 0.85. CN 1.15μg/mL SD 0.55. p<0.0001). Moreover, fetal triglycerid levels were significantly increased in IUGR (IUGR 16.7mg/dL SD 7.58. CN 56.5mg/dL SD 49.92. p-value after log transformation =0.0008)and apo C-III0 was highly correlated to fetal triglyceride levels (rho=0.694). CONCLUSION Using mass spectrometric approaches we successfully developed an IUGR specific proteome signature derived from human umbilical cord blood samples. Most interesting the deglycosylated form of the apolipoprotein C-III (apoC-III0) was found to be significantly increased in IUGR and thus might lead to reduced triglyceride catabolism. This observation is in agreement with the known observation of triglyceride levels being increased in IUGR fetuses. Our results indicate that subtle alterations in protein glycosylation need to be considered for improving our understanding of the pathomechanisms in IUGR.