Sickle cell disease in adults

This issue of eMedRef provides information to clinicians on sickle cell disease in adults

Adolescent Psychiatry Volume developmental and clinical studies

-515

Glycogen, not dehydration or lipids, limits winter survival of side-blotched lizards (Uta stansburiana)

Climate change is causing winters to become milder (less cold and shorter). Recent studies of overwintering ectotherms have suggested that warmer winters increase metabolism and decrease winter survival and subsequent fecundity. Energetic constraints (insufficient energy stores) have been hypothesized as the cause of winter mortality but have not been tested explicitly. Thus, alternative sources of mortality, such as winter dehydration, cannot be ruled out. By employing an experimental design that compared the energetics and water content of lizards that died naturally during laboratory winter with those that survived up to the same point but were then sacrificed, we attempt to distinguish among multiple possible causes of mortality. We test the hypothesis that mortality is caused by insufficient energy stores in the liver, abdominal fat bodies, tail or carcass or through excessive water loss. We found that lizards that died naturally had marginally greater mass loss, lower water content, and less liver glycogen remaining than living animals sampled at the same time. Periodically moistening air during winter reduced water loss, but this did not affect survival, calling into question dehydration as a cause of death. Rather, our results implicate energy limitations in the form of liver glycogen, but not lipids, as the primary cause of mortality in overwintering lizards. When viewed through a lens of changing climates, our results suggest that if milder winters increase the metabolic rate of overwintering ectotherms, individuals may experience greater energetic demands. Increased energy use during winter may subsequently limit individual survival and possibly even impact population persistence

Parallel workflow for high-throughput (>1,000 samples/day) quantitative analysis of human insulin-like growth factor 1 using mass spectrometric immunoassay.

Insulin-like growth factor 1 (IGF1) is an important biomarker for the management of growth hormone disorders. Recently there has been rising interest in deploying mass spectrometric (MS) methods of detection for measuring IGF1. However, widespread clinical adoption of any MS-based IGF1 assay will require increased throughput and speed to justify the costs of analyses, and robust industrial platforms that are reproducible across laboratories. Presented here is an MS-based quantitative IGF1 assay with performance rating of >1,000 samples/day, and a capability of quantifying IGF1 point mutations and posttranslational modifications. The throughput of the IGF1 mass spectrometric immunoassay (MSIA) benefited from a simplified sample preparation step, IGF1 immunocapture in a tip format, and high-throughput MALDI-TOF MS analysis. The Limit of Detection and Limit of Quantification of the resulting assay were 1.5 μg/L and 5 μg/L, respectively, with intra- and inter-assay precision CVs of less than 10%, and good linearity and recovery characteristics. The IGF1 MSIA was benchmarked against commercially available IGF1 ELISA via Bland-Altman method comparison test, resulting in a slight positive bias of 16%. The IGF1 MSIA was employed in an optimized parallel workflow utilizing two pipetting robots and MALDI-TOF-MS instruments synced into one-hour phases of sample preparation, extraction and MSIA pipette tip elution, MS data collection, and data processing. Using this workflow, high-throughput IGF1 quantification of 1,054 human samples was achieved in approximately 9 hours. This rate of assaying is a significant improvement over existing MS-based IGF1 assays, and is on par with that of the enzyme-based immunoassays. Furthermore, a mutation was detected in ∼1% of the samples (SNP: rs17884626, creating an A→T substitution at position 67 of the IGF1), demonstrating the capability of IGF1 MSIA to detect point mutations and posttranslational modifications

IGF1 methods comparison.

<p>A) Scatter plot. B) Difference plot.</p

Mass spectra from a normal individual (A) and an individual with a single nucleotide polymorphism (B). m/z values refer to the average mass [M+H]⁺.

<p>A) Native IGF1 was detected (observed 7649.99 m/z, theoretical 7649.71) along with a putative glycosylated variant (labeled with *; observed 8346.90 m/z, theoretical unknown). B) Both IGF1 (observed 7649.75 m/z, theoretical 7649.71) and IGF1 A67T (observed 7679.75 m/z, theoretical 7679.71) were detected as well as their respective putative glycosylated variants (observed 8349.35 m/z, theoretical unknown and observed 8379.15 m/z, theoretical unknown).</p

Spiking recovery.

<p>Spiking recovery.</p

An example of a standard curve for the IGF1 MSIA.

<p>Plotted are the peak area ratios of IGF1/LR3-IGF1 over the standards concentrations. Solid line: linear fit with R² = 0.99, SEE =  0.69. Dotted lines: 95% prediction intervals. The average r² for the twelve standard curves was 0.98 and the range was 0.97–0.99.</p