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ABSTRACT: BACKGROUND:Immunoassays for 1α,25-dihydroxyvitamin D [1α,25(OH)(2)D] lack analytical specificity. We characterized the cross-reactivity of an anti-1α,25(OH)(2)D antibody with purified vitamin D metabolites and used these data to map the chemical features of 1α,25(OH)(2)D that are important for antibody binding. Additionally, we hypothesized that when combined with isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS), antibody cross-reactivity could be used to semiselectively enrich for structurally similar metabolites of vitamin D in a multiplexed assay.METHODS:Sample preparation consisted of immunoaffinity enrichment with a solid-phase anti-1α,25(OH)(2)D antibody and derivatization. Analytes were quantified with LC-MS/MS. Supplementation and recovery studies were performed for 11 vitamin D metabolites. We developed a method for simultaneously quantifying 25(OH)D(2), 25(OH)D(3), 24,25(OH)(2)D(3), 1α,25(OH)(2)D(2), and 1α,25 (OH)(2)D(3) that included deuterated internal standards for each analyte.RESULTS:The important chemical features of vitamin D metabolites for binding to the antibody were (a) native orientation of the hydroxyl group on carbon C3 in the A ring, (b) the lack of substitution at carbon C4 in the A ring, and (c) the overall polarity of the vitamin D metabolite. The multiplexed method had lower limits of quantification (20% CV) of 0.2 μg/L, 1.0 μg/L, 0.06 μg/L, 3.4 pg/mL, and 2.8 pg/mL for 25(OH)D(2), 25(OH)D(3), 24,25(OH)(2)D(3), 1α,25(OH)(2)D(2), and 1α,25 (OH)(2)D(3), respectively. Method comparisons to 3 other LC-MS/MS methods yielded an r(2) value >0.9, an intercept less than the lower limit of quantification, and a slope statistically indistinguishable from 1.0.CONCLUSIONS:LC-MS/MS can be used to characterize antibody cross-reactivity, a conclusion supported by our multiplexed assay for 5 vitamin D metabolites with immunoenrichment in a targeted metabolomic assay.
Clinical Chemistry 09/2012; · 7.91 Impact Factor
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ABSTRACT: Iron is critical in multiple aspects of CNS development, but its role in neurodevelopment--the ability of iron deficiency to alter normal development--is difficult to dissociate from the effects of anemia. We developed a novel dietary restriction model in the rat that allows us to study the effects of iron deficiency in the absence of severe anemia. Using a combination of auditory brainstem response analyses (ABR) and electron microscopy, we identified an unexpected impact of nonanemic iron deficiency on axonal diameter and neurofilament regulation in the auditory nerve. These changes are associated with altered ABR latency during development. In contrast to models of severe iron deficiency with anemia, we did not find consistent or prolonged defects in myelination. Our data demonstrate that iron deficiency in the absence of anemia disrupts normal development of the auditory nerve and results in altered conduction velocity.
Journal of Neuroscience 04/2012; 32(14):5010-5. · 7.11 Impact Factor
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ABSTRACT: The implementation of mass spectrometry to measure serum 25-hydroxyvitamin D [25(OH)D] concentrations has led to concerns regarding the measurement and reporting of the C3-epimer of 25-hydroxyvitamin D(3) [3-epi-25(OH)D(3)], for which there is a near-total lack of data regarding its clinical significance.
We developed a chromatographic method to resolve (>90%) 3-epi-25(OH)D(3) from 25(OH)D(3) using a pentafluorophenyl propyl chromatographic column. Using LC-MS/MS, we determined the serum concentrations of 25(OH)D(3) and 3-epi-25(OH)D(3) in 626 patients aged 3 days to 94 years undergoing routine vitamin D testing.
Comparison between DiaSorin RIA and the new LC-MS/MS method for total 25(OH)D had acceptable agreement. Our data indicate an increase in 25(OH)D(3) rather than a reduction in epimer concentration. An average of 3.3 ng/ml of 3-epi-25(OH)D(3) was detected in adolescents and adults. Inclusion of 3-epi-25(OH)D(3) in the total 25(OH)D(3) concentration resulted in 9% (<1 year) and 3% (1 to 94 years) potential misclassification of patients as vitamin D sufficient.
The new LC-MS/MS method is capable of chromatographically separating 25(OH)D(3) and 3-epi-25(OH)D(3). It was used to confirm that the contribution of 3-epi-25OHD(3) to total 25OHD(3) concentrations decreases with age in infants and is detectable in adults.
Clinica chimica acta; international journal of clinical chemistry 01/2012; 413(1-2):203-6. · 2.54 Impact Factor
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ABSTRACT: Despite their widespread use, the performance of delta check rules is rarely evaluated because errors are rare and lack a gold standard for detection. In this study we used a simulation-based approach to compare strategies for empirically defining criteria for univariate delta checks, and assessed the performance of these rules for detecting mislabeled specimens in 2 inpatient populations.
We performed simulations using historical laboratory test results by randomly sampling pairs of specimens successively drawn from the same patient or two different patients. We evaluated the performance of delta check rules using a variety of thresholds, including those currently in use in our laboratory.
Mean corpuscular volume had the highest positive predictive value for specimen mislabeling, and produced the fewest false positives. Conversely, rules using other laboratory tests had considerably poorer performance. Several of the "best guess" thresholds historically used in our laboratory, notably those for potassium and anion gap, were predicted to have extremely low yields. In addition, rule performance was not consistent between the two patient populations.
The low yield of delta checks based on any single analyte should prompt careful evaluation of their practical utility. Furthermore, our results indicate that it may not be possible to generalize delta rules across institutions.
Clinica chimica acta; international journal of clinical chemistry 10/2011; 412(21-22):1973-7. · 2.54 Impact Factor
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ABSTRACT: Mass spectrometry is an analytic technique with high specificity and a growing presence in laboratory medicine. Various types of mass spectrometers are being used in an increasing number of clinical laboratories around the world, and, as a result, significant improvements in assay performance are occurring rapidly in areas such as toxicology, endocrinology, and biochemical genetics. This review serves as a basic introduction to mass spectrometry, its uses, and associated challenges in the clinical laboratory and ends with a brief discussion of newer methods with the greatest potential for clinical diagnostics.
American Journal of Clinical Pathology 10/2011; 136(4):609-16. · 2.60 Impact Factor
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ABSTRACT: We investigated the use of serum samples from BD Vacutainer rapid serum tubes (RSTs; BD, Franklin Lakes, NJ) to reduce undetermined interferences contributing to false-positive immunoassay results in heparin plasma samples. Patients being evaluated for suspected myocardial infarction had specimens drawn into an RST in addition to the standard lithium-heparin plasma separator tube (PST). We measured 28 separate analytes in both specimens using immunoassay, electrochemical, and spectrophotometric methods. Higher results were observed in some PST specimens tested for troponin I, creatine kinase-MB isoenzyme, human chorionic gonadotropin, and thyroid-stimulating hormone. These discrepancies were investigated by repeating analyses after recentrifugation of both specimens. Reanalysis gave results for the PST specimens that were lower and agreed well with initial results from RSTs, suggesting false-positive rates of 10.8% for troponin I and about 2% for each of the other 3 analytes. Overall, specimens collected in RSTs had fewer false-positive immunoassay results than specimens collected in plasma separator tubes.
American Journal of Clinical Pathology 08/2011; 136(2):325-9. · 2.60 Impact Factor
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ABSTRACT: 1α,25-dihydroxy vitamin D [1,25(OH)(2)D] is the active metabolite of vitamin D. Antibody-based detection methods lack specificity, but when combined with isotope dilution/ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry, immunoextraction provides an attractive method for 1,25(OH)(2)D. We developed a method for simultaneous quantification of 1,25(OH)(2)D(2) and 1,25(OH)(2)D(3) with a 4.6-min instrument cycle time. Results are available 36 h after sample preparation begins.
Sample preparation consisted of protein precipitation, immunoextraction with solid-phase anti-1,25(OH)(2)D antibody, and derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione. Analytes were resolved using reversed-phase UPLC and quantified using positive ion electrospray ionization-tandem mass spectrometry. We used hexadeuterated 1,25(OH)(2)D(3) and 1,25(OH)(2)D(2) as internal standards and performed method comparisons against the DiaSorin RIA and an LC-MS/MS method available at a reference laboratory.
1,25(OH)(2)D(3) intraassay and interassay imprecision was 5.6% and 8.0% (120 pmol/L) and 8.7% and 13% (48 pmol/L). Limits of detection and quantification were 1.5 pmol/L and 3.0 pmol/L, respectively. 1,25(OH)(2)D(2) intraassay and interassay imprecision was 8.7% and 11% (186 pmol/L) and 11% and 13% (58 pmol/L). Limits of detection and quantification were both 1.5 pmol/L. Comparison with RIA had a proportional bias of 0.75, constant bias of -4.1, and Pearson correlation (r(2)) of 0.31. Comparison with a reference LC-MS/MS assay had a proportional bias of 0.89, constant bias of 3.7, and r(2) of 0.88.
Protein precipitation with antibody-based extraction is effective for sample preparation before LC-MS/MS analysis of derivatized 1,25(OH)(2)D. This method appears to have improved specificity over a clinically used RIA with low imprecision and limits of detection.
Clinical Chemistry 07/2011; 57(9):1279-85. · 7.91 Impact Factor
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ABSTRACT: Neurogenesis involves the proliferation of multipotent neuroepithelial stem cells followed by differentiation into lineage-restricted neural precursor cells (NPCs) during the embryonic period. Interestingly, these progenitor cells express robust levels of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that regulates expression of genes important for growth regulation, and xenobiotic metabolism. Upon binding 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a pervasive environmental contaminant and potent AhR ligand, AhR, is activated and disrupts gene expression patterns to produce cellular toxicity. Because of its widespread distribution in the brain during critical proliferative phases of neurogenesis, it is conceivable that AhR participates in NPC expansion. Therefore, this study tested the hypothesis that AhR activation by TCDD disrupts signaling events that regulate NPC proliferation. The C17.2 NPC line served as a model system to (1) assess whether NPCs are targets for TCDD-induced neurotoxicity and (2) characterize the effects of TCDD on NPC proliferation. We demonstrated that C17.2 NPCs express an intact AhR signaling pathway that becomes transcriptionally active after TCDD exposure. (3)H-thymidine and alamar blue reduction assays indicated that TCDD suppresses NPC proliferation in a concentration-dependent manner without the loss of cell viability. Cell cycle distribution analysis by flow cytometry revealed that TCDD-induced growth arrest results from an impaired G1 to S cell cycle transition. Moreover, TCDD exposure altered p27( kip1) and cyclin D1 cell cycle regulatory protein expression levels consistent with a G1 phase arrest. Initial studies in primary NPCs isolated from the ventral forebrain of embryonic mice demonstrated that TCDD reduced cell proliferation through a G1 phase arrest, corroborating our findings in the C17.2 cell line. Together, these observations suggest that the inappropriate or sustained activation of AhR by TCDD during neurogenesis can interfere with signaling pathways that regulate neuroepithelial stem cell/NPC proliferation, which could adversely impact final cell number in the brain and lead to functional impairments.
Stem cells and development 02/2011; 20(2):313-26. · 4.15 Impact Factor
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ABSTRACT: It is well acknowledged from observations in humans that iron deficiency during pregnancy can be associated with a number of developmental problems in the newborn and developing child. Due to the obvious limitations of human studies, the stage during gestation at which maternal iron deficiency causes an apparent impairment in the offspring remains elusive. In order to begin to understand the time window(s) during pregnancy that is/are especially susceptible to suboptimal iron levels, which may result in negative effects on the development of the fetus, we developed a rat model in which we were able to manipulate and monitor the dietary iron intake during specific stages of pregnancy and analyzed the developing fetuses. We established four different dietary-feeding protocols that were designed to render the fetuses iron deficient at different gestational stages. Based on a functional analysis that employed Auditory Brainstem Response measurements, we found that maternal iron restriction initiated prior to conception and during the first trimester were associated with profound changes in the developing fetus compared to iron restriction initiated later in pregnancy. We also showed that the presence of iron deficiency anemia, low body weight, and changes in core body temperature were not defining factors in the establishment of neural impairment in the rodent offspring.Our data may have significant relevance for understanding the impact of suboptimal iron levels during pregnancy not only on the mother but also on the developing fetus and hence might lead to a more informed timing of iron supplementation during pregnancy.
PLoS ONE 01/2011; 6(3):e17483. · 4.09 Impact Factor
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ABSTRACT: Considerably less attention has been given to understanding the cellular components of gliogenesis in the telencephalon when compared to neuronogenesis, despite the necessity of normal glial cell formation for neurological function. Early proposals of exclusive ventral oligodendrocyte precursor cell (OPC) generation have been challenged recently with studies revealing the potential of the dorsal telencephalon to also generate oligodendrocytes. The identification of OPCs generated from multiple regions of the developing telencephalon, together with the need of the embryonic telencephalon to provide precursor cells for oligodendrocytes as well as astrocytes in ventral and dorsal areas, raises questions concerning the identity of the precursor cell populations capable of generating macroglial subtypes during multiple developmental windows and in differing locations.
We have identified progenitor populations in the ventral and dorsal telencephalon restricted to the generation of astrocytes and oligodendrocytes. We further demonstrate that the dorsal glial progenitor cells can be generated de novo from the dorsal telencephalon and we demonstrate their capacity for in vivo production of both myelin-forming oligodendrocytes and astrocytes upon transplantation.
Based on our results we offer a unifying model of telencephalic gliogenesis, with the generation of both oligodendrocytes and astrocytes from spatially separate, but functionally similar, glial restricted populations at different developmental times in the dorsal and ventral CNS.
BMC Developmental Biology 02/2007; 7:33. · 2.79 Impact Factor
