Citations

Abstract Background: Newborn screening for congenital adrenal hyperplasia (CAH) involves measurement of 17α-hydroxyprogesterone (17-OHP), usually by immunoassay. Because this testing has been characterized by high false-positive rates, we developed a steroid profiling method that uses liquid chromatography–tandem mass spectrometry (LC-MS/MS) to measure 17-OHP, androstenedione, and cortisol simultaneously in blood spots. Methods: Whole blood was eluted from a 4.8-mm (3/16-inch) dried-blood spot by an aqueous solution containing the deuterium-labeled internal standard d8-17-OHP. 17-OHP, androstenedione, and cortisol were extracted into diethyl ether, which was subsequently evaporated and the residue dissolved in LC mobile phase. This extract was injected into a LC-MS/MS equipped with pneumatically assisted electrospray. The steroids were quantified in the selected-reaction monitoring mode by use of peak areas in reference to the stable-isotope-labeled internal standard. We analyzed 857 newborn blood spots, including 14 blood spots of confirmed CAH cases and 101 of false-positive cases by conventional screening. Results: Intra- and interassay CVs for 17-OHP were 7.2–20% and 3.9–18%, respectively, at concentrations of 2, 30, and 50 μg/L. At a cutoff for 17-OHP of 12.5 μg/L and a cutoff of 3.75 for the sum of peak areas for 17-OHP and androstenedione divided by the peak area for cortisol, 86 of the 101 false-positive samples were within reference values by LC-MS/MS, whereas the 742 normal and 14 true-positive results obtained by conventional screening were correctly classified. Conclusion: Steroid profiling in blood spots can identify false-positive results obtained by conventional newborn screening for CAH.

Abstract Background: Multiple endocrine neoplasia type 2 (MEN2) is a cancer syndrome with well-characterized causative mutations. Missense mutations in ∼15 codons of the RET gene have been linked to disease phenotypes in the vast majority of cases. These missense mutations range from very simple single nucleotide base changes to more numerous changes at a given codon; they therefore are often tested for by more than one DNA-based diagnostic method. We developed and evaluated a Pyrosequencing™ technology-based approach for MEN2 mutation testing that allows both simple and complex mutations to be analyzed on one platform. Methods: Archived DNA from peripheral blood of patients referred to the Mayo Clinic Molecular Genetics laboratory for MEN2 testing was selected. One to all of codons 609, 611, 618, 620, 630, 634, 768, 804, and 918 were analyzed by Pyrosequencing technology to match the original analysis of each patient. Template PCRs were set up using an automated liquid handler; the subsequent post-PCR preparation step was performed manually, and the sequencing was performed by a PSQ 96 instrument. Samples were tested in batch sizes expected to occur routinely. Results: We analyzed samples from 217 patients who previously tested negative for MEN2 and 230 patients who previously tested positive, for a total of 1449 sequencing reactions. One discrepant result was found (100% concordant for negatives and 99.6% concordant for positives). A total of 37 unique mutations or alterations of unknown significance were analyzed. Conclusion: Pyrosequencing technology offers an accurate, nonisotopic, simple, and rapid method for the analysis of DNA from patients suspected of having MEN2.

AbstractBackground: Quantification of plasma free metanephrines is usually accomplished by HPLC with electrochemical detection, but sample preparation is labor-intensive and time-consuming, run times are long, and interfering substances sometimes obscure the relevant peaks. The aim of this study was to develop a sensitive and specific LC-MS/MS method for plasma free metanephrines.Methods: After solid-phase extraction, chromatographic separation of normetanephrine (NMN) and metanephrine (MN) was accomplished by use of a cyano analytical column. NMN, MN, d3-NMN, and d3-MN positive ions were detected in the multiple-reaction monitoring mode using the specific transitions m/z 166→134, 180→148, 169→137, and 183→151, respectively, with an atmospheric pressure chemical ionization source.Results: Multiple calibration curves exhibited consistent linearity and reproducibility. Interassay imprecision values (CV; n = 20) for NMN at 0.64, 1.9, and 2.7 nmol/L were 6.6%, 7.8%, and 13%, respectively. Interassay CV for MN at 0.60, 1.2, and 2.1 nmol/L (n = 20) were 9.2%, 6.8%, and 9.8%, respectively. The mean recoveries of NMN and MN relative to the internal standard were 100% and 96%, respectively. The assays were linear between 0.20 and 10.0 nmol/L. Deming regression of HPLC and LC-MS/MS results yielded slopes of 0.93 (95% confidence interval, 0.89–0.98) and 0.89 (0.85–0.93) and y-intercepts of −0.16 and 0.03 nmol/L for NMN (n = 132) and MN (n = 92), respectively.Conclusions: This novel LC-MS/MS approach provides a precise, rapid, and specific alternative method to HPLC for the quantification of the low nanomolar concentrations of free metanephrines in plasma.

The relationship between iron status and atherosclerosis has long been a topic of debate in the literature. Despite more than 25 years of research, there is no consensus regarding a causal relationship. To date, the vast majority of studies have focused on iron burden with respect to a hypothesized role in the onset and progression of coronary artery disease. However, the effect of iron in the coronary arterial system may differ mechanistically and therefore clinically from its effect in the peripheral arterial system. This review will summarize the biochemical, pathologic, animal, and clinical research data with respect to iron and atherosclerosis. This background will be expanded upon to provide insights into ongoing studies and paths for future investigations into the role of iron and peripheral arterial disease.

Pregnancy-associated plasma protein A (PAPPA) is a metzincin superfamily metalloproteinase in the insulin-like growth factor (IGF) system. PAPPA increases IGF bioavailability and mitogenic effectiveness in vitro through regulated cleavage of IGF-binding protein 4 (IGFBP4). To determine its function in vivo, we generated PAPPA-null mice by gene targeting. Mice homozygous for targeted disruption of the PAPPA gene were viable but 60% the size of wild-type littermates at birth. The impact of the mutation was exerted during the early embryonic period prior to organogenesis, resulting in proportional dwarfism. PAPPA, IGF2 and IGFBP4 transcripts co-localized in wild-type embryos, and expression of IGF2 and IGFBP4 mRNA was not altered in PAPPA-deficient embryos. However,IGFBP4 proteolytic activity was completely lacking in fibroblasts derived from PAPPA-deficient embryos, and IGFBP4 effectively inhibited IGF-stimulated mitogenesis in these cells. These results provide the first direct evidence that PAPPA is an essential growth regulatory factor in vivo, and suggest a novel mechanism for regulated IGF bioavailability during early fetal development.

Background and Purpose— Incomplete platelet inhibition by aspirin (aspirin resistance) may be a reason for stroke recurrence in some patients. 11-Dehydrothromboxane B2 (11-DTB2) is a stable thromboxane A2 metabolite that reflects in vivo platelet activation. This pilot study was intended to evaluate the reproducibility of urinary 11-DTB2 over time and to look for evidence of aspirin resistance. Methods— All subjects were screened for the African American Antiplatelet Stroke Prevention Study (AAASPS) 7 to 90 days after noncardioembolic cerebral infarction. Of 83 subjects with at least 1 urine sample, 52 were enrolled in AAASPS (randomized to blinded treatment with aspirin 650 mg/d or ticlopidine 500 mg/d), and 31 were enrolled in an open-label antiplatelet therapy cohort. Subjects were followed up for 2 years, with 11-DTB2 measurements scheduled at baseline and 6, 12, and 24 months. Vascular events were cerebral infarction, myocardial infarction, or vascular death. Results— Despite considerable individual up or down fluctuations, the median 11-DTB2 change did not significantly differ from zero in any of the subgroups. However, in 6 subjects with a 4-fold decrease in aspirin dose from 1300 to 325 or 81 mg/d, the 11-DTB2 level increased from 611 to 1881 pg/mg creatinine ( P =0.06). Vascular events occurred in 7 of 61 aspirin-treated subjects, and 11-DTB2 levels did not correlate with the events. Conclusions— Fluctuations in urinary 11-DTB2 after cerebral infarction in blacks do not correlate with changes in aspirin doses, except perhaps when the dose changes by a factor of 4 or more. A larger study is needed to look further for aspirin resistance.

Mechanical unloading of the heart with a left ventricular assist device (LVAD) significantly decreases mortality in patients with heart failure. Moreover, it provides a human model to define the critical regulatory genes governing myocardial remodeling in response to significant reductions in wall stress. Statistical analysis of a gene expression library of 19 paired human heart samples harvested at the time of LVAD implant and again at explant revealed a set of 22 genes that were downregulated and 85 genes that were upregulated in response to mechanical unloading with a false discovery rate of less than 1%. The analysis revealed a high percentage of genes involved in the regulation of vascular networks including neuropilin-1 (a VEGF receptor), FGF9, Sprouty1, stromal-derived factor 1, and endomucin. Taken together these findings suggest that mechanical unloading alters the regulation of vascular organization and migration in the heart. In addition to vascular signaling networks, GATA-4 binding protein, a critical mediator of myocyte hypertrophy, was significantly downregulated following mechanical unloading. In summary, these findings may have important implications for defining the role of mechanical stretch and load on autocrine/paracrine signals directing vascular organization in the failing human heart and the role of GATA-4 in orchestrating reverse myocardial remodeling. This unbiased gene discovery approach in paired human heart samples has the potential to provide critical clues to the next generation of therapeutic treatments aimed at heart failure.