Neonatal screening for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in The Netherlands: the importance of enzyme analysis to ascertain true MCAD deficiency.

The outcome was determined of population-wide neonatal screening for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency using tandem mass spectrometry (MS/MS) in The Netherlands, between October 2003 and September 2005. Prospective population-wide neonatal screening for MCAD deficiency was performed in the northern part of The Netherlands. In newborns with blood octanoylcarnitine (C(8:0)) concentrations > or =0.3 micromol/L, clinical and laboratory follow-up was initiated, including MCAD enzymatic measurements which played a decisive role. In a 2-year period, 66 216 newborns were investigated for MCAD deficiency and follow-up was initiated in 28 newborns. True-positives (n = 14) were identified based upon MCAD enzyme activity <50%, measured with hexanoyl-CoA as substrate. The observed prevalence of MCAD deficiency was 1/6600 (95% CI: 1/4100-1/17 400). In addition to an elevated C(8:0) concentration, a C(8:0)/C(10:0) molar ratio >5.0 turned out to differentiate between false-positives and true-positives. Measurement of MCAD activity using phenylpropionyl-CoA as a substrate further discriminated between newborns with MCAD deficiency and so-called mild MCAD deficiency. To summarize, neonatal screening for MCAD deficiency in the northern part of The Netherlands resulted in the predicted number of affected newborns. Measurement of MCAD activity in leukocytes or lymphocytes using phenylpropionyl-CoA as a substrate can be regarded as the gold standard to diagnose MCAD deficiency upon initial positive screening test results.

Plasma TGFbeta level in rats after hemithoracic irradiation.

Changes in TGF-beta plasma levels were observed 18 weeks after hemithoracic irradiation in rats. This coincides with an increase in the breathing frequency. being most pronounced between 22 and 28 weeks after irradiation. The correlation suggests a potential role of the circulating TGF-beta in the monitoring of localized radiation-induced lung injury.

Quantification of transforming growth factor-beta in biological material using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct.

Transforming growth factor-beta (TGF-beta), a multifunctional cytokine, can be quantified by a variety of bioassays or immunoassays. One of the disadvantages of these techniques is that they require sample purification to remove components that interfere with the TGF-beta signal. In the current study the feasibility of quantifying TGF-beta in complex biological fluids directly with a recently developed bioassay was examined. This assay is based on the ability of TGF-beta to induce plasminogen activator inhibitor-1 (PAI-1) expression. Mature TGF-beta binds to the receptors of mink lung epithelial cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct (PAI/L), resulting in a dose-dependent increase of luciferase activity. Specificity for TGF-beta was proven by treatment of the samples with neutralizing antibodies. The sensitivity and the intraassay precision are comparable to the ELISA. It is demonstrated, however, that, unlike the ELISA, a purification step by, e.g., acid-ethanol extraction prior to the PAI/L assay, is not required. This not only simplifies the assay but also reduces the minimal sample volume and allows to discriminate between latent and mature TGF-beta. The present study furthermore provides insight in the critical steps for accurate TGF-beta determination, which include careful blood collection and sample handling (storage and preparation). With this protocol TGF-beta has been quantified in human plasma, rat plasma, rat saliva, tissue extracts from rat lung, and in culture medium of TGF-beta-producing cells.

Feasibility of measuring radiation-induced DNA double strand breaks and their repair by pulsed field gel electrophoresis in freshly isolated cells from the mouse RIF-1 tumor.

PURPOSE: To examine the technical feasibility of pulsed field gel electrophoresis (PFGE) as a predictive assay for the radioresponsiveness of tumors. Induction and repair of DNA double strand breaks (DSBs) in a freshly prepared cell suspension from a RIF-1 tumor (irradiated ex vivo) was compared with DSB induction and repair in exponentially growing RIF-1 cells in culture (irradiated in vitro). METHODS AND MATERIALS: A murine RIF-1 tumor grown in vivo was digested, and cells were exposed to x-rays (ex vivo) at doses of 1 to 75 Gy. DNA damage was measured using CHEF (clamped homogeneous electric fields) electrophoresis. Repair kinetics were studied at 37 degrees C for 4 h after irradiation. Radiosensitivity was determined by clonogenic assay, and cell cycle distributions by flow cytometry. For comparison, a trypsinized suspension of exponentially growing RIF-1 cells in vitro was run parallel with each ex vivo experiment. RESULTS: Induction of DSBs, expressed as % DNA extracted from the plug, was similar in the in vitro and ex vivo irradiated cells. Compared to repair rates in vitro cultured RIF-1 cells, repair kinetics in a freshly prepared cell suspension from the tumor were decreased, unrelated to differences in radiosensitivity. Differences in repair could not be explained by endogenous DNA degradation, nor by influences of enzymes used for digestion of the tumor. A lower plating efficiency and differences in ploidy (as revealed by flow cytometry) were the only reproducible differences between in vivo and in vitro grown cells that may explain the differences in repair kinetics. CONCLUSIONS: The current results do not support the idea that PFGE is a technique robust enough to be a predictive assay for the radiosensitivity of tumor cells.